@article{dutton_hinman_kim_attayek_maurer_sims_allbritton_2021, title={Hyperglycemia minimally alters primary self-renewing human colonic epithelial cells while TNF alpha-promotes severe intestinal epithelial dysfunction}, volume={13}, ISSN={["1757-9708"]}, DOI={10.1093/intbio/zyab008}, abstractNote={Abstract}, number={6}, journal={INTEGRATIVE BIOLOGY}, author={Dutton, Johanna S. and Hinman, Samuel S. and Kim, Raehyun and Attayek, Peter J. and Maurer, Mallory and Sims, Christopher S. and Allbritton, Nancy L.}, year={2021}, month={Jun}, pages={139–152} }
 @article{hinman_huling_wang_wang_bretherton_deforest_allbritton_2021, title={Magnetically-propelled fecal surrogates for modeling the impact of solid-induced shear forces on primary colonic epithelial cells}, volume={276}, ISSN={["1878-5905"]}, DOI={10.1016/j.biomaterials.2021.121059}, abstractNote={The colonic epithelium is continuously exposed to an array of biological and mechanical stimuli as its luminal contents are guided over the epithelial surface through regulated smooth muscle contraction. In this report, the propulsion of solid fecal contents over the colonic epithelium is recapitulated through noninvasive actuation of magnetic agarose hydrogels over primary intestinal epithelial cultures, in contrast to the vast majority of platforms that apply shear forces through liquid microflow. Software-controlled magnetic stepper motors enable experimental control over the frequency and velocity of these events to match in vivo propulsive contractions, while the integration of standardized well plate spacing facilitates rapid integration into existing assay pipelines. The application of these solid-induced shear forces did not deleteriously affect cell monolayer surface coverage, viability, or transepithelial electrical resistance unless the device parameters were raised to a 50× greater contraction frequency and 4× greater fecal velocity than those observed in healthy humans. At a frequency and velocity that is consistent with average human colonic motility, differentiation of the epithelial cells into absorptive and goblet cell phenotypes was not affected. Protein secretion was modulated with a two-fold increase in luminal mucin-2 secretion and a significant reduction in basal interleukin-8 secretion. F-actin, zonula occludens-1, and E-cadherin were each present in their proper basolateral locations, similar to those of static control cultures. While cellular height was unaffected by magnetic agarose propulsion, several alterations in lateral morphology were observed including decreased circularity and compactness, and an increase in major axis length, which align with surface epithelial cell morphologies observed in vivo and may represent early markers of luminal exfoliation. This platform will be of widespread utility for the investigation of fecal propulsive forces on intestinal physiology, shedding light on how the colonic epithelium responds to mechanical cues.}, journal={BIOMATERIALS}, author={Hinman, Samuel S. and Huling, Jennifer and Wang, Yuli and Wang, Hao and Bretherton, Ross C. and deForest, Cole A. and Allbritton, Nancy L.}, year={2021}, month={Sep} }
 @article{mestril_kim_hinman_gomez_allbritton_2021, title={Stem/Proliferative and Differentiated Cells within Primary Murine Colonic Epithelium Display Distinct Intracellular Free Ca2+ Signal Codes}, ISSN={["2192-2659"]}, DOI={10.1002/adhm.202101318}, abstractNote={Abstract}, journal={ADVANCED HEALTHCARE MATERIALS}, author={Mestril, Sebastian and Kim, Raehyun and Hinman, Samuel S. and Gomez, Shawn M. and Allbritton, Nancy L.}, year={2021}, month={Sep} }
 @article{kim_attayek_wang_furtado_tamayo_sims_allbritton_2020, title={An in vitro intestinal platform with a self-sustaining oxygen gradient to study the human gut/microbiome interface}, volume={12}, ISSN={["1758-5090"]}, DOI={10.1088/1758-5090/ab446e}, abstractNote={An oxygen gradient formed along the length of colonic crypts supports stem-cell proliferation at the normoxic crypt base while supporting obligate anaerobe growth in the anoxic colonic lumen. Primary human colonic epithelial cells derived from human gastrointestinal stem cells were cultured within a device possessing materials of tailored oxygen permeability to produce an oxygen-depleted luminal (0.8% ± 0.1% O2) and oxygen-rich basal (11.1% ± 0.5% O2) compartment. This oxygen difference created a stable oxygen gradient across the colonic epithelial cells which remained viable and properly polarized. Facultative and obligate anaerobes Lactobacillus rhamnosus, Bifidobacterium adolescentis, and Clostridium difficile grew readily within the luminal compartment. When formed along the length of an in vitro crypt, the oxygen gradient facilitated cell compartmentalization within the crypt by enhancing confinement of the proliferative cells to the crypt base. This platform provides a simple system to create a physiological oxygen gradient across an intestinal mimic while simultaneously supporting anaerobe co-culture.}, number={1}, journal={BIOFABRICATION}, author={Kim, Raehyun and Attayek, Peter J. and Wang, Yuli and Furtado, Kathleen L. and Tamayo, Rita and Sims, Christopher E. and Allbritton, Nancy L.}, year={2020}, month={Jan} }
 @article{labelle_zhang_armistead_allbritton_2020, title={Assay and Isolation of Single Proliferating CD4+Lymphocytes Using an Automated Microraft Array Platform}, volume={67}, ISSN={["1558-2531"]}, DOI={10.1109/TBME.2019.2956081}, abstractNote={Objective: While T lymphocytes have been employed as a cancer immunotherapy, the development of effective and specific T-cell-based therapeutics remains challenging. A key obstacle is the difficulty in identifying T cells reactive to cancer-associated antigens. The objective of this research was to develop a versatile platform for single cell analysis and isolation that can be applied in immunology research and clinical therapy development. Methods: An automated microscopy and cell sorting system was developed to track the proliferative behavior of single-cell human primary CD4+ lymphocytes in response to stimulation using allogeneic lymphoblastoid feeder cells. Results: The system identified single human T lymphocytes with a sensitivity of 98% and specificity of 99% and possessed a cell collection efficiency of 86%. Time-lapse imaging simultaneously tracked 4,534 alloreactive T cells on a single array; 19% of the arrayed cells formed colonies of ≥2 cells. From the array, 130 clonal colonies were isolated and 7 grew to colony sizes of >10,000 cells, consistent with the known proliferative capacity of T cells in vitro and their tendency to become exhausted with prolonged stimulation. The isolated colonies underwent ELISA assay to detect interferon-γ secretion and Sanger sequencing to determine T cell receptor β sequences with a 100% success rate. Conclusion: The platform is capable of both identification and isolation of proliferative T cells in an automated manner. Significance: This novel technology enables the identification of TCR sequences based on T cell proliferation which is expected to speed the development of future cancer immunotherapies.}, number={8}, journal={IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING}, author={LaBelle, Cody A. and Zhang, Raymond J. and Armistead, Paul M. and Allbritton, Nancy L.}, year={2020}, pages={2166–2175} }
 @article{nowotarski_attayek_allbritton_2020, title={Automated platform for cell selection and separation based on four-dimensional motility and matrix degradation}, volume={145}, ISSN={["1364-5528"]}, DOI={10.1039/c9an02224d}, abstractNote={An automated platform to separate single cells based on matrix invasion was implemented on a microraft array to track inheritability of this complex phenotype.}, number={7}, journal={ANALYST}, author={Nowotarski, Hannah L. and Attayek, Peter J. and Allbritton, Nancy L.}, year={2020}, month={Apr}, pages={2731–2742} }
 @misc{labelle_massaro_cortes-llanos_sims_allbritton_2020, title={Image-Based Live Cell Sorting}, volume={39}, ISSN={["1879-3096"]}, DOI={10.1016/j.tibtech.2020.10.006}, abstractNote={Technologies capable of cell separation based on cell images provide powerful tools enabling cell selection criteria that rely on spatially or temporally varying properties. Image-based cell sorting (IBCS) systems utilize microfluidic or microarray platforms, each having unique characteristics and applications. The advent of IBCS marks a new paradigm in which cell phenotype and behavior can be explored with high resolution and tied to cellular physiological and omics data, providing a deeper understanding of single-cell physiology and the creation of cell lines with unique properties. Cell sorting guided by high-content image information has far-reaching implications in biomedical research, clinical medicine, and pharmaceutical development.}, number={6}, journal={TRENDS IN BIOTECHNOLOGY}, author={LaBelle, Cody A. and Massaro, Angelo and Cortes-Llanos, Belen and Sims, Christopher E. and Allbritton, Nancy L.}, year={2020}, month={Jun}, pages={613–623} }
 @article{smiddy_disalvo_allbritton-king_allbritton_2020, title={Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies}, volume={145}, ISSN={["1364-5528"]}, DOI={10.1039/d0an00030b}, abstractNote={A magnetic, microwell-based plate was established enabling automated collection of colony biopsy fragments into known locations for immunofluorescence assays.}, number={7}, journal={ANALYST}, author={Smiddy, Nicole M. and DiSalvo, Matthew and Allbritton-King, Jules D. and Allbritton, Nancy L.}, year={2020}, month={Apr}, pages={2649–2660} }
 @article{wheeler_vu_einstein_disalvo_ahmed_van nostrand_shishkin_jin_allbritton_yee_2020, title={Pooled CRISPR screens with imaging on microraft arrays reveals stress granule-regulatory factors}, volume={17}, ISSN={["1548-7105"]}, DOI={10.1038/s41592-020-0826-8}, abstractNote={Genetic screens using pooled CRISPR-based approaches are scalable and inexpensive, but restricted to standard readouts, including survival, proliferation and sortable markers. However, many biologically relevant cell states involve cellular and subcellular changes that are only accessible by microscopic visualization, and are currently impossible to screen with pooled methods. Here we combine pooled CRISPR–Cas9 screening with microraft array technology and high-content imaging to screen image-based phenotypes (CRaft-ID; CRISPR-based microRaft followed by guide RNA identification). By isolating microrafts that contain genetic clones harboring individual guide RNAs (gRNA), we identify RNA-binding proteins (RBPs) that influence the formation of stress granules, the punctate protein–RNA assemblies that form during stress. To automate hit identification, we developed a machine-learning model trained on nuclear morphology to remove unhealthy cells or imaging artifacts. In doing so, we identified and validated previously uncharacterized RBPs that modulate stress granule abundance, highlighting the applicability of our approach to facilitate image-based pooled CRISPR screens. CRISPR-based microraft followed by guide RNA identification (CRaft-ID) combines microraft arrays, microscopy and CRISPR–Cas9 technology for high-content image-based phenotyping. CRaft-ID was used to identify proteins involved in stress granule formation.}, number={6}, journal={NATURE METHODS}, author={Wheeler, Emily C. and Vu, Anthony Q. and Einstein, Jaclyn M. and DiSalvo, Matthew and Ahmed, Noorsher and Van Nostrand, Eric L. and Shishkin, Alexander A. and Jin, Wenhao and Allbritton, Nancy L. and Yee, Gene W.}, year={2020}, month={Jun}, pages={636-+} }
 @misc{gallion_anttila_abraham_proctor_allbritton_2020, title={Preserving single cells in space and time for analytical assays}, volume={122}, ISSN={["1879-3142"]}, DOI={10.1016/j.trac.2019.115723}, abstractNote={Analytical assays performed within clinical laboratories influence roughly 70% of all medical decisions by facilitating disease detection, diagnosis, and management. Both in clinical and academic research laboratories, single-cell assays permit measurement of cell diversity and identification of rare cells, both of which are important in the understanding of disease pathogenesis. For clinical utility, the single-cell assays must be compatible with the clinical workflow steps of sample collection, sample transportation, pre-analysis processing, and single-cell assay; therefore, it is paramount to preserve cells in a state that resembles that in vivo rather than measuring signaling behaviors initiated in response to stressors such as sample collection and processing. To address these challenges, novel cell fixation (and more broadly, cell preservation) techniques incorporate programmable fixation times, reversible bond formation and cleavage, chemoselective reactions, and improved analyte recovery. These technologies will further the development of individualized, precision therapies for patients to yield improved clinical outcomes.}, journal={TRAC-TRENDS IN ANALYTICAL CHEMISTRY}, author={Gallion, Luke A. and Anttila, Matthew M. and Abraham, David H. and Proctor, Angela and Allbritton, Nancy L.}, year={2020}, month={Jan} }
 @article{proctor_allbritton_2019, title={"Fix and assay": separating in-cellulo sphingolipid reactions from analytical assay in time and space using an aldehyde-based fixative}, volume={144}, ISSN={["1364-5528"]}, DOI={10.1039/c8an01353e}, abstractNote={Fixation of fluorescent sphingolipid-loaded cells enables cell metabolism and assay readout to be separated in time and space.}, number={3}, journal={ANALYST}, author={Proctor, Angela and Allbritton, Nancy L.}, year={2019}, month={Feb}, pages={961–971} }
 @article{disalvo_smiddy_allbritton_2019, title={Automated sensing and splitting of stem cell colonies on microraft arrays}, volume={3}, ISSN={["2473-2877"]}, DOI={10.1063/1.5113719}, abstractNote={Human induced pluripotent stem cells (hiPSCs) are widely used for disease modeling, tissue engineering, and clinical applications. Although the development of new disease-relevant or customized hiPSC lines is of high importance, current automated hiPSC isolation technologies rely largely on the fluorescent labeling of cells, thus limiting the cell line development from many applications. The objective of this research was to develop a platform for high-throughput hiPSC cytometry and splitting that utilized a label-free cell sensing approach. An image analysis pipeline utilizing background subtraction and standard deviation projections was implemented to detect hiPSC colonies from bright-field microscopy data. The pipeline was incorporated into an automated microscopy system coupling quad microraft cell-isolation arrays, computer-based vision, and algorithms for smart decision making and cell sorting. The pipeline exhibited a hiPSC detection specificity of 98% and a sensitivity of 88%, allowing for the successful tracking of growth for hundreds of microcolonies over 7 days. The automated platform split 170 mother colonies from a microarray within 80 min, and the harvested daughter biopsies were expanded into viable hiPSC colonies suitable for downstream assays, such as polymerase chain reaction (PCR) or continued culture. Transmitted light microscopy offers an alternative, label-free modality for isolating hiPSCs, yet its low contrast and specificity for adherent cells remain a challenge for automation. This novel approach to label-free sensing and microcolony subsampling with the preservation of the mother colony holds the potential for hiPSC colony screening based on a wide range of properties including those measurable only by a cell destructive assay.}, number={3}, journal={APL BIOENGINEERING}, author={DiSalvo, Matthew and Smiddy, Nicole M. and Allbritton, Nancy L.}, year={2019}, month={Sep} }
 @misc{wang_kim_sims_allbritton_2019, title={Building a Thick Mucus Hydrogel Layer to Improve the Physiological Relevance of In Vitro Primary Colonic Epithelial Models}, volume={8}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2019.07.009}, abstractNote={Human colonic mucosa is covered with a mucus layer several hundred microns in thickness, which serves as a barrier physically separating the underlying epithelium from bacteria, and hindering the movement of molecules derived from food and microbiota.1Johansson M.E.V. et al.Proc Natl Acad Sci U S A. 2011; 108: 4659-4665Crossref PubMed Scopus (709) Google Scholar, 2Hansson G.C. et al.Curr Opin Microbiol. 2012; 15: 57-62Crossref PubMed Scopus (252) Google Scholar, 3Werlang C. et al.Nat Rev Mater. 2019; 4: 134-145Crossref Scopus (19) Google Scholar Despite its importance, a thick, continuous mucus layer that mimics in vivo functions has not been achieved in in vitro colonic epithelial models constructed from primary intestinal epithelial cells. The organoid “mini-gut” model possesses goblet cells, but mucus is secreted and accumulated within the organoids’ inaccessible lumen making the mucus properties difficult to quantify.4Sato T. et al.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (1602) Google Scholar, 5Dedhia P.H. et al.Gastroenterology. 2016; 150: 1098-1112Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar Prior monolayer models exposed the tissue luminal surface by culturing primary cells on scaffolds, but the mucus layer was very thin and diffuse and unable to act as a physiologic barrier.6In J. et al.Cell Mol Gastroenterol Hepatol. 2016; 2: 48-62.e3Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 7Moon C. et al.Mucosal Immunol. 2014; 7: 818-828Crossref PubMed Scopus (133) Google Scholar In this study, we report strategies to generate a thick mucus layer and demonstrate its function act as a physiological barrier to bacteria and toxins. An air-liquid interface (ALI) similar to that used for respiratory cell culture8Prunieras M. et al.J Investig Dermatol. 1983; 81: S28-S33Abstract Full Text PDF PubMed Scopus (353) Google Scholar was tested based on the hypothesis that overlying aqueous medium with convective mixing might dilute the mucus as it formed. Removal of the fluid might then enable accumulation of a thick, condensed mucus. Human colonic epithelial monolayers were differentiated for 5 days as either a submerged or as an ALI culture (Supplementary Figure 1A). A contiguous mucus layer with irregular thickness (71–381 μm) was observed in the ALI culture by immunofluorescence (Supplementary Figure 1B, D) but was absent from the submerged culture. The mucus layer appeared to fully cover the epithelium when viewed by scanning electron microscopy (Supplementary Figure 1C). Additionally, the mucus layer served as a barrier to 1-μm red fluorescent beads and green fluorescent protein–expressing Escherichia coli separating them from the epithelium (Supplemental Figure 1D). The ALI culture was modified by adding an endogenous intestinal hormone, vasoactive intestinal peptide (VIP),9Schwartz C.J. et al.J Clin Invest. 1974; 54: 536-544Crossref PubMed Scopus (268) Google Scholar, 10Wu X. et al.PLoS One. 2015; 10e0125225PubMed Google Scholar to mediate the luminal water homeostasis with a goal of supporting hydrated mucus layer of uniform thickness (Figure 1A). VIP stimulated the apical water secretion in a dose-dependent manner with an effective dose 50 of 210 pg/mL (Figure 1B). When the monolayers were differentiated under ALI culture with VIP, a layer of liquid (42 mg/cm2) accumulated on the apical side within 24 hours and was maintained during 5 days of differentiation. The ALI culture with VIP significantly increased the number of goblet cells (Figure 1C; Supplementary Figure 2A, B). The apical mucus volume increased in quantity over time and by day 5 a slippery hydrogel could be lifted off the epithelium (Figure 1D). The thickness of the bacteria-separating mucus layer was readily adjusted by the duration of ALI culture (Figure 1E, F; Supplemental Figure 2C). The thickness of the mucus was even across the surface (302 ± 28 μm), likely due to its ability to flow and redistribute as a result of its water content unlike that in the ALI culture without VIP. The mucus effectively separated 1-μm red fluorescent beads from the underlying epithelium (Figure 2G, Supplemental Figure 2E). Scanning electron microscopy images additionally demonstrated a confluent layer of uninterrupted mucus upon which microbes could be visualized (Supplemental Figure 2D). The mucus layer contained other expected constituents including Muc5AC, TFF3, and AGR2 (Figure 1H; Supplementary Figure 2G). As the single mucus layer acted as barrier to microbes, its properties resembled that of the compact or microbe-impermeant layer observed in vivo. However, vigorous washing removed the mucus layer entirely, a property most closely associated with the diffuse mucus layer bordering the luminal contents in vivo (Supplementary Figure 2E). Overlaying the mucus layer with a mixture of 0.02- and 5-μm beads suggested the presence of a single layer (Supplementary Figure 2F).Figure 2Effect of Clostridium difficile toxin A and Escherichia coli (EC) on human colonic epithelium in the absence or presence of the VIP-enhanced mucus layer. (A) Cell-culture schematic for panels B and C. (B) Permeability and (C) IL-8 secretion of the epithelium after 4-hour exposure to toxin A. (D) Cell culture schematic for panels E, F, and G. (E–G) Production of inflammatory cytokines after a 24-hour co-culture of green fluorescent protein (GFP)–expressing EC, epithelium, and peripheral blood mononuclear cells (PBMC): (E) tumor necrosis factor alpha (TNF-α), (F) IL-6, and (G) IL-1β. Unpaired t test: **P < .005; #not statistically significant. n = 3 samples per condition. N.S., not significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To demonstrate the physiological barrier function of the mucus, intestinal epithelial monolayers were incubated with Clostridium difficile toxin A (Figure 2A) or E coli (Figure 2D) in the absence or presence of a mucus layer. In the absence of a mucus layer, toxin A disrupted apical F-actin structures and zonula occludens-1 tight junctions, diminished epithelial barrier function, and elicited epithelial-cell interleukin (IL)-8 secretion by 4 hours (Supplementary Figure 3A). In the presence of a mucus layer, the epithelial cells were protected from the destructive effects of toxin at 4 hours (Figure 2B, C). Although by 8 hours, the tissue demonstrated signs of toxin exposure (Supplementary Figure 3B, C). The in vitro mucus layer also served as a physical barrier to segregate E coli from the epithelium and underlying white blood cells for up to 24 hours with elimination of cytokine production (tumor necrosis factor alpha, IL-6, and IL-1β) in the presence of the mucus layer (Figure 2E–G). These results demonstrate that the mucus layer successfully slowed toxin movement, delaying intestinal damage, and acted as a physical barrier for bacteria, delaying or eliminating the immune response and emulating in vivo conditions. The authors thank the Scott Magness Laboratory for kindly providing human colon specimens. The authors also thank Shee-Hwan Hwang and Mallory Maurer for technical assistance. The human-derived colonic epithelial stem cells were expanded in stem medium (SM) (formulation is listed in Supplementary Table 1) using a previously published protocol.1Wang Y. et al.Cell Mol Gastroenterol Hepatol. 2017; 4: 165-182Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 2Wang Y. et al.Cell Mol Gastroenterol Hepatol. 2018; 5: 113-130Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 3Wang Y. et al.Anal Chem. 2018; 90: 11523-11530Crossref PubMed Scopus (12) Google Scholar Twelve-well Transwell inserts possessing a porous membrane (0.4-μm pore size) (#3460; Corning, Corning, NY) were coated with 1 vol% Matrigel in ice cold phosphate-buffered saline at 37°C overnight and rinsed with phosphate-buffered saline. Cells were plated as described previously in EM.3Wang Y. et al.Anal Chem. 2018; 90: 11523-11530Crossref PubMed Scopus (12) Google Scholar The medium was switched to differentiation medium (DM) after 5 days.3Wang Y. et al.Anal Chem. 2018; 90: 11523-11530Crossref PubMed Scopus (12) Google Scholar In the submerged culture, 1-mL DM was added in the apical reservoir and 2 mL was added in the basal reservoir. In the air-liquid interface culture, the medium in the apical reservoir was completely aspirated, 1-mL DM or DM vasoactive intestinal peptide (VIP) (DM containing 330-ng/mL VIP [#AS-22872; AnaSpec, Fremont, CA]) was added to the basal reservoir. DM or DM-VIP was changed daily. Cells were cultured for 10 days before assay. The cells and mucus layer were fixed with Carnoy’s solution (ethanol 6:acetic acid 3:chloroform 1, v/v/v) at 4°C for 2 hours, dehydrated in ethanol, paraffin-embedded, sectioned, and stained with anti-Muc2 antibody (#sc-15334, 200×; Santa Cruz Biotechnology, Dallas, TX) and Hoechst 33342 (#62249; ThermoFisher)2Wang Y. et al.Cell Mol Gastroenterol Hepatol. 2018; 5: 113-130Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar or antibodies against TFF3 (#PA557279, 100×; ThermoFisher), AGR2 (#PA534517, 100×; ThermoFisher), RELM-β (#PA561896, 100×; ThermoFisher) and MUC5AC (#MA512178, 100×; ThermoFisher).4Madsen J. et al.J Histochem Cytochem. 2007; 55: 505-513Crossref PubMed Scopus (150) Google Scholar, 5Zhao F. et al.Dev Biol. 2010; 338: 270-279Crossref PubMed Scopus (153) Google Scholar, 6Barnes S.L. et al.J Immunol. 2007; 179: 7012-7020Crossref PubMed Scopus (28) Google Scholar For scanning electron microscopy imaging, the tissues were fixed with Carnoy’s solution or glyoxal at 4°C for 2 hours, dehydrated in a graded ethanol dried with a critical point dryer (Tousimis Semidri PVT-3, Rockville, MD), coated with 10-nm metal by a sputter coater (Cressington 108, Watford, England, UK), and inspected by scanning electron microscopy (FEI Quanta 200 ESEM; FEI, Hillsboro, OR). To demonstrate the barrier capability, the cells were stained with Hoechst 33342 (2 μg/mL) from basal side and then green fluorescent protein–expressing Escherichia coli (EC) (0.5 mL) (#25922GFP; ATCC, Manassas, VA) at 200 million colony-forming units (CFU)/mL, or red fluorescent beads (1-μm diameter) (#F13083; ThermoFisher) at a density of 108 beads/mL was added to the apical reservoir. Twenty minutes after seeding, the Transwell insert were imaged by confocal microscopy. A mixture (20 μL) of natural Clostridium difficile toxin A protein (12 μg/mL) (#ab123999; Abcam, Cambridge, UK) and FITC-dextran (5 mg/mL) (#FD40S; Sigma-Aldrich, St Louis, MO) was added to the apical monolayer side. Hanks' balanced salt solution with calcium/magnesium, 10% fetal bovine serum (FBS), and 10-mM HEPES was added to the basal compartment. Samples (150 μL) were collected from the basal compartment at 2, 4, 8, and 24 hours. The fluorescence intensity of the collected samples was used to calculate the percent permeability. The cells were fixed in ethanol and stained with phalloidin (#R37110; ThermoFisher) and zonula occludens-1 antibody (#21773-1-AP; Proteintech Group, Rosemont, IL). Fresh human peripheral blood mononuclear cells (Physician's Plasma Alliance, Johnson City, TN) were suspended in RPMI containing 10% FBS and 100-μg/mL gentamicin at 2.86 million cells/mL. GFP-expressing EC (200 million CFU/mL) were washed and suspended in 0.2-mL RPMI with FBS and gentamicin (100 μg/mL).7Parlesak A. et al.Scand J Immunol. 2004; 60: 477-485Crossref PubMed Scopus (65) Google Scholar GFP-expressing EC (20 million CFU, 20 μL) was added to the apical epithelial side. Peripheral blood mononuclear cells (1.43 million, 500 μL) were added to the basal reservoir. After 24 hours, the basal reservoir media reservoir was collected, centrifuged (5000 rpm, 6 minutes), aliquoted, and stored at –20°C. The concentrations of cytokines (interleukin [IL]-8, IL-6, IL-1β, and tumor necrosis factor alpha) were determined using enzyme-linked immunosorbent assay kits (ThermoFisher, Waltham, MA) (n = 3) . Advanced DMEM/F-12, Dulbecco's Modified Eagle Medium/Ham's F-12; DM, differentiation medium; EGF, epidermal growth factor; EM, expansion medium; WRN, Wnt-3A, R-spondin 3, Noggin; PGE2, prostaglandin E2; SM, stem medium; VIP, vasoactive intestinal peptide.}, number={4}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Wang, Y. and Kim, R. and Sims, C. E. and Allbritton, N. L.}, year={2019}, pages={653-+} }
 @article{speer_wang_fallon_smith_allbritton_2019, title={Evaluation of human primary intestinal monolayers for drug metabolizing capabilities}, volume={13}, ISSN={["1754-1611"]}, DOI={10.1186/s13036-019-0212-1}, abstractNote={Abstract}, number={1}, journal={JOURNAL OF BIOLOGICAL ENGINEERING}, author={Speer, Jennifer E. and Wang, Yuli and Fallon, John K. and Smith, Philip C. and Allbritton, Nancy L.}, year={2019}, month={Nov} }
 @article{speer_gunasekara_wang_fallon_attayek_smith_sims_allbritton_2019, title={Molecular transport through primary human small intestinal monolayers by culture on a collagen scaffold with a gradient of chemical cross-linking}, volume={13}, ISSN={["1754-1611"]}, DOI={10.1186/s13036-019-0165-4}, abstractNote={The luminal surface of the small intestine is composed of a monolayer of cells overlying a lamina propria comprised of extracellular matrix (ECM) proteins. The ECM provides a porous substrate critical for nutrient exchange and cellular adhesion. The enterocytes within the epithelial monolayer possess proteins such as transporters, carriers, pumps and channels that participate in the movement of drugs, metabolites, ions and amino acids and whose function can be regulated or altered by the properties of the ECM. Here, we characterized expression and function of proteins involved in transport across the human small intestinal epithelium grown on two different culture platforms. One strategy employs a conventional scaffolding method comprised of a thin ECM film overlaying a porous membrane while the other utilizes a thick ECM hydrogel placed on a porous membrane. The thick hydrogel possesses a gradient of chemical cross-linking along its length to provide a softer substrate than that of the ECM film-coated membrane while maintaining mechanical stability.The monolayers on both platforms possessed goblet cells and abundant enterocytes and were impermeable to Lucifer yellow and fluorescein-dextran (70 kD) indicating high barrier integrity. Multiple transporter proteins were present in both primary-cell culture formats at levels similar to those present in freshly isolated crypts/villi; however, expression of breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the monolayers on the conventional scaffold was substantially less than that on the gradient cross-linked scaffold and freshly isolated crypts/villi. Monolayers on the conventional scaffold failed to transport the BCRP substrate prazosin while cells on the gradient cross-linked scaffold successfully transported this drug to better mimic the properties of in vivo small intestine.The results of this comparison highlight the need to create in vitro intestinal transport platforms whose characteristics mimic the in vivo lamina propria in order to accurately recapitulate epithelial function.}, journal={JOURNAL OF BIOLOGICAL ENGINEERING}, author={Speer, Jennifer E. and Gunasekara, Dulan B. and Wang, Yuli and Fallon, John K. and Attayek, Peter J. and Smith, Philip C. and Sims, Christopher E. and Allbritton, Nancy L.}, year={2019}, month={Apr} }
 @article{hinman_wang_allbritton_2019, title={Photopatterned Membranes and Chemical Gradients Enable Scalable Phenotypic Organization of Primary Human Colon Epithelial Models}, volume={91}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.9b04217}, abstractNote={Biochemical gradients across the intestinal epithelium play a major role in governing intestinal stem cell compartmentalization, differentiation dynamics, and organ-level self-renewal. However, scalable platforms that recapitulate the architecture and gradients present in vivo are absent. We present a platform in which individually addressable arrays of chemical gradients along the intestinal crypt long axis can be generated, enabling scalable culture of primary in vitro colonic epithelial replicas. The platform utilizes standardized well plate spacing, maintains access to basal and luminal compartments, and relies on a photopatterned porous membrane to act as diffusion windows while supporting the in vitro crypts. Simultaneous fabrication of 3875 crypts over a single membrane was developed. Growth factor gradients were modeled and then experimentally optimized to promote long-term health and self-renewal of the crypts which were assayed in situ by confocal fluorescence microscopy. The cultured in vitro crypt arrays successfully recapitulated the architecture and luminal-to-basal phenotypic polarity observed in vivo. Furthermore, known signaling regulators (e.g., butyrate and DAPT) produced measurable and predictable effects on the organized cell compartments, each decreasing crypt proliferation in the basal regions to negligible values. This platform is readily adaptable to the screening of tissue from individual patients to assay the impact of food and bacterial metabolites and/or drugs on colonic crypt dynamics. Importantly, the cassette is compatible with a wide range of sensing/detection modalities, and the developed fabrication methods should find applications for other cell and tissue types.}, number={23}, journal={ANALYTICAL CHEMISTRY}, author={Hinman, Samuel S. and Wang, Yuli and Allbritton, Nancy L.}, year={2019}, month={Dec}, pages={15240–15247} }
 @misc{dutton_hinman_kim_wang_allbritton_2019, title={Primary Cell-Derived Intestinal Models: Recapitulating Physiology}, volume={37}, ISSN={["1879-3096"]}, DOI={10.1016/j.tibtech.2018.12.001}, abstractNote={The development of physiologically relevant intestinal models fueled by breakthroughs in primary cell-culture methods has enabled successful recapitulation of key features of intestinal physiology. These advances, paired with engineering methods, for example incorporating chemical gradients or physical forces across the tissues, have yielded ever more sophisticated systems that enhance our understanding of the impact of the host microbiome on human physiology as well as on the genesis of intestinal diseases such as inflammatory bowel disease and colon cancer. In this review we highlight recent advances in the development and usage of primary cell-derived intestinal models incorporating monolayers, organoids, microengineered platforms, and macrostructured systems, and discuss the expected directions of the field.}, number={7}, journal={TRENDS IN BIOTECHNOLOGY}, author={Dutton, Johanna S. and Hinman, Samuel S. and Kim, Raehyun and Wang, Yuli and Allbritton, Nancy L.}, year={2019}, month={Jul}, pages={744–760} }
 @article{proctor_wang_lawrence_allbritton_2019, title={Selection and optimization of enzyme reporters for chemical cytometry}, volume={622}, ISSN={["0076-6879"]}, DOI={10.1016/bs.mie.2019.02.023}, abstractNote={Chemical cytometry, sensitive analytical measurements of single cells, reveals inherent heterogeneity of cells within a population which is masked or averaged out when using bulk analysis techniques. A particular challenge of chemical cytometry is the development of a suitable reporter or probe for the desired measurement. These reporters must be sufficiently specific for measuring the desired process; possess a lifetime long enough to accomplish the measurement; and have the ability to be loaded into single cells. This chapter details our approach to rationally design and improve peptide substrates as reporters of enzyme activity utilizing chemical cytometry. This method details the iterative approach used to design, characterize, and identify a peptidase-resistant peptide reporter which acts as a kinase substrate within intact cells. Small-scale, rationally designed peptide libraries are generated to rapidly and economically screen candidate reporter peptides for substrate suitability and peptidase resistance. Also detailed are strategies to characterize and validate the designed reporters by determining kinetic parameters, intracellular substrate specificity, resistance to degradation by intracellular peptidases, and behavior within lysates and intact cells.}, journal={CHEMICAL AND SYNTHETIC BIOLOGY APPROACHES TO UNDERSTAND CELLULAR FUNCTIONS - PT B}, author={Proctor, Angela and Wang, Qunzhao and Lawrence, David S. and Allbritton, Nancy L.}, year={2019}, pages={221–248} }
 @article{gunasekara_speer_wang_nguyen_reed_smiddy_parker_fallon_smith_sims_et al._2018, title={A Monolayer of Primary Colonic Epithelium Generated on a Scaffold with a Gradient of Stiffness for Drug Transport Studies}, volume={90}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.8b02845}, abstractNote={Animal models are frequently used for in vitro physiologic and drug transport studies of the colon, but there exists significant pressure to improve assay throughput as well as to achieve tighter control of experimental variables than can be achieved with animals. Thus, development of a primary in vitro colonic epithelium cultured as high resistance with transport protein expression and functional behavior similar to that of a native colonic would be of enormous value for pharmaceutical research. A collagen scaffold, in which the degree of collagen cross-linking was present as a gradient, was developed to support the proliferation of primary colonic cells. The gradient of cross-linking created a gradient in stiffness across the scaffold, enabling the scaffold to resist deformation by cells. mRNA expression and quantitative proteomic mass spectrometry of cells growing on these surfaces as a monolayer suggested that the transporters present were similar to those in vivo. Confluent monolayers acted as a barrier to small molecules so that drug transport studies were readily performed. Transport function was evaluated using atenolol (a substrate for passive paracellular transport), propranolol (a substrate for passive transcellular transport), rhodamine 123 (Rh123, a substrate for P-glycoprotein), and riboflavin (a substrate for solute carrier transporters). Atenolol was poorly transported with an apparent permeability ( Papp) of <5 × 10-7 cm s-1, while propranolol demonstrated a Papp of 9.69 × 10-6 cm s-1. Rh123 was transported in a luminal direction ( Papp,efflux/ Papp,influx = 7) and was blocked by verapamil, a known inhibitor of P-glycoprotein. Riboflavin was transported in a basal direction, and saturation of the transporter was observed at high riboflavin concentrations as occurs in vivo. It is anticipated that this platform of primary colonic epithelium will find utility in drug development and physiological studies, since the tissue possesses high integrity and active transporters and metabolism similar to that in vivo.}, number={22}, journal={ANALYTICAL CHEMISTRY}, author={Gunasekara, Dulan B. and Speer, Jennifer and Wang, Yuli and Nguyen, Daniel L. and Reed, Mark I. and Smiddy, Nicole M. and Parker, Joel S. and Fallon, John K. and Smith, Philip C. and Sims, Christopher E. and et al.}, year={2018}, month={Nov}, pages={13331–13340} }
 @article{wang_kim_hwang_dutton_sims_allbritton_2018, title={Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform}, volume={90}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.8b02835}, abstractNote={In vitro models of the human intestinal epithelium derived from primary stem cells are much needed for the study of intestinal immunology in health and disease. Here, we describe an intestinal monolayer cultured on a porous membrane with accessible basal and apical surfaces for assay of intestinal cytokine production in response to stimuli. The system was composed of a differentiated, confluent epithelial monolayer derived from human primary stem cells obtained from small or large intestine. Interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) were the most abundant inflammatory cytokines produced by the intestinal epithelium. The epithelium from all five tested regions of the intestine preferentially secreted into the apical reservoir of the monolayer, with a 26-fold greater concentration of IL-8 present in the apical reservoir of the colonic monolayer relative to that in the basal reservoir. Upon application of tumor-necrosis factor α (TNF-α) to the basal surface of the colonic monolayer, the IL-8 concentration significantly increased in the basal, but not the apical, reservoir. A dose-dependent elevation of IL-8 in the basal reservoir was observed for TNF-α-stimulation of the monolayer but not for an organoid-based platform. To demonstrate the utility of the monolayer system, 88 types of dietary metabolites or compounds were screened for their ability to modulate IL-8 production in the basal reservoir of the intestinal monolayer in the absence or presence of TNF-α. No dietary metabolite or compound caused an increase in IL-8 in the basal reservoir in the absence of TNF-α. After addition of TNF-α to the monolayer, two compounds (butyrate and gallic acid) suppressed IL-8 production, suggesting their potential anti-inflammatory effects, whereas the dietary factor forskolin significantly increased IL-8 production. These results demonstrate that the described human-intestinal-monolayer platform has the potential for assays and screening of metabolites and compounds that alter the inflammatory response of the intestine.}, number={19}, journal={ANALYTICAL CHEMISTRY}, author={Wang, Yuli and Kim, Raehyun and Hwang, Shee-Hwan J. and Dutton, Johanna and Sims, Christopher E. and Allbritton, Nancy L.}, year={2018}, month={Oct}, pages={11523–11530} }
 @misc{wang_kim_hinman_zwarycz_magness_allbritton_2018, title={Bioengineered systems and designer matrices that recapitulate the intestinal stem cell niche}, volume={5}, number={3}, journal={Cellular and Molecular Gastroenterology and Hepatology}, author={Wang, Y. L. and Kim, R. and Hinman, S. S. and Zwarycz, B. and Magness, S. T. and Allbritton, N. L.}, year={2018}, pages={440-} }
 @article{disalvo_harris_kantesaria_pena_allbritton-king_cole_allbritton_2018, title={Characterization of Tensioned PDMS Membranes for Imaging Cytometry on Microraft Arrays}, volume={90}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.8b00176}, abstractNote={Polydimethylsiloxane (PDMS) membranes can act as sensing elements, barriers, and substrates, yet the low rigidity of the elastomeric membranes can limit their practical use in devices. Microraft arrays rely on a freestanding PDMS membrane as a substrate for cell arrays used in imaging cytometry and cellular isolation. However, the underlying PDMS membrane deforms under the weight of the cell media, making automated analytical microscopy (and thus cytometry and cell isolation) challenging. Here we report the development of microfabrication strategies and physically motivated mathematical modeling of membrane deformation of PDMS microarrays. Microraft arrays were fabricated with mechanical tension stored within the PDMS substrate. These membranes deformed 20× less than that of arrays fabricated using prior methods. Modeling of the deformation of pretensioned arrays using linear membrane theory yielded ≤15% error in predicting the array deflection and predicted the impact of cure temperatures up to 120 °C. A mathematical approach was developed to fit models of microraft shape to sparse real-world shape measurements. Automated imaging of cells on pretensioned microarrays using the focal planes predicted by the model produced high quality fluorescence images of cells, enabling accurate cell area quantification (<4% error) at increased speed (13×) relative to conventional methods. Our microfabrication method and simplified, linear modeling approach is readily applicable to control the deformation of similar membranes in MEMs devices, sensors, and microfluidics.}, number={7}, journal={ANALYTICAL CHEMISTRY}, author={DiSalvo, Matthew and Harris, Daniel M. and Kantesaria, Saurin and Pena, Alexis N. and Allbritton-King, Jules D. and Cole, Jacqueline H. and Allbritton, Nancy L.}, year={2018}, month={Apr}, pages={4792–4800} }
 @article{wang_kim_gunasekara_reed_disalvo_nguyen_bultman_sims_magness_allbritton_2018, title={Formation of Human Colonic Crypt Array by Application of Chemical Gradients Across a Shaped Epithelial Monolayer}, volume={5}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2017.10.007}, abstractNote={<h3>Background & Aims</h3> The successful culture of intestinal organoids has greatly enhanced our understanding of intestinal stem cell physiology and enabled the generation of novel intestinal disease models. Although of tremendous value, intestinal organoid culture systems have not yet fully recapitulated the anatomy or physiology of the in vivo intestinal epithelium. The aim of this work was to re-create an intestinal epithelium with a high density of polarized crypts that respond in a physiologic manner to addition of growth factors, metabolites, or cytokines to the basal or luminal tissue surface as occurs in vivo. <h3>Methods</h3> A self-renewing monolayer of human intestinal epithelium was cultured on a collagen scaffold microfabricated with an array of crypt-like invaginations. Placement of chemical factors in either the fluid reservoir below or above the cell-covered scaffolding created a gradient of that chemical across the growing epithelial tissue possessing the in vitro crypt structures. Crypt polarization (size of the stem/proliferative and differentiated cell zones) was assessed in response to gradients of growth factors, cytokines, and bacterial metabolites. <h3>Results</h3> Chemical gradients applied to the shaped human epithelium re-created the stem/proliferative and differentiated cell zones of the in vivo intestine. Short-chain fatty acids applied as a gradient from the luminal side confirmed long-standing hypotheses that butyrate diminished stem/progenitor cell proliferation and promoted differentiation into absorptive colonocytes. A gradient of interferon-γ and tumor necrosis factor-α significantly suppressed the stem/progenitor cell proliferation, altering crypt formation. <h3>Conclusions</h3> The in vitro human colon crypt array accurately mimicked the architecture, luminal accessibility, tissue polarity, cell migration, and cellular responses of in vivo intestinal crypts.}, number={2}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Wang, Yuli and Kim, Raehyun and Gunasekara, Dulan B. and Reed, Mark I. and DiSalvo, Matthew and Nguyen, Daniel L. and Bultman, Scott J. and Sims, Christopher E. and Magness, Scott T. and Allbritton, Nancy L.}, year={2018}, pages={113–130} }
 @article{fadero_gerbich_rana_suzuki_disalvo_schaefer_heppert_boothby_goldstein_peifer_et al._2018, title={LITE microscopy: Tilted light-sheet excitation of model organisms offers high resolution and low photobleaching}, volume={217}, ISSN={["1540-8140"]}, DOI={10.1083/jcb.201710087}, abstractNote={Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.}, number={5}, journal={JOURNAL OF CELL BIOLOGY}, author={Fadero, Tanner C. and Gerbich, Therese M. and Rana, Kishan and Suzuki, Aussie and DiSalvo, Matthew and Schaefer, Kristina N. and Heppert, Jennifer K. and Boothby, Thomas C. and Goldstein, Bob and Peifer, Mark and et al.}, year={2018}, month={May}, pages={1869–1882} }
 @article{fayer_gilliland_ramsey_allbritton_waters_2018, title={N-Gemini peptides: cytosolic protease resistance via N-terminal dimerization of unstructured peptides}, volume={54}, ISSN={["1364-548X"]}, DOI={10.1039/c7cc06819k}, abstractNote={N-Terminal dimerization is a synthetically straight-forward strategy to provide protease resistance to unstructured peptides while maintaining their biological function.}, number={2}, journal={CHEMICAL COMMUNICATIONS}, author={Fayer, Effrat L. and Gilliland, William M., Jr. and Ramsey, J. Michael and Allbritton, Nancy L. and Waters, Marcey L.}, year={2018}, month={Jan}, pages={204–207} }
 @article{bhatt_gunasekara_speer_reed_pena_midkiff_magness_bultman_allbritton_redinbo_2018, title={Nonsteroidal Anti-Inflammatory Drug -Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells}, volume={4}, ISSN={["2373-8227"]}, DOI={10.1021/acsinfecdis.7b00139}, abstractNote={The intestinal epithelium provides a critical barrier that separates the gut microbiota from host tissues. Nonsteroidal anti-inflammatory drugs (NSAIDs) are efficacious analgesics and antipyretics and are among the most frequently used drugs worldwide. In addition to gastric damage, NSAIDs are toxic to the intestinal epithelium, causing erosions, perforations, and longitudinal ulcers in the gut. Here, we use a unique in vitro human primary small intestinal cell monolayer system to pinpoint the intestinal consequences of NSAID treatment. We found that physiologically relevant doses of the NSAID diclofenac (DCF) are cytotoxic because they uncouple mitochondrial oxidative phosphorylation and generate reactive oxygen species. We also find that DCF induces intestinal barrier permeability, facilitating the translocation of compounds from the luminal to the basolateral side of the intestinal epithelium. The results we outline here establish the utility of this novel platform, representative of the human small intestinal epithelium, to understand NSAID toxicity, which can be applied to study multiple aspects of gut barrier function including defense against infectious pathogens and host-microbiota interactions.}, number={1}, journal={ACS INFECTIOUS DISEASES}, author={Bhatt, Aadra P. and Gunasekara, Dulan B. and Speer, Jennifer and Reed, Mark I. and Pena, Alexis N. and Midkiff, Bentley R. and Magness, Scott T. and Bultman, Scott J. and Allbritton, Nancy L. and Redinbo, Matthew R.}, year={2018}, month={Jan}, pages={46–52} }
 @article{wang_gunasekara_reed_disalvo_bultman_sims_magness_allbritton_2017, title={A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium}, volume={128}, ISSN={["1878-5905"]}, DOI={10.1016/j.biomaterials.2017.03.005}, abstractNote={The human small intestinal epithelium possesses a distinct crypt-villus architecture and tissue polarity in which proliferative cells reside inside crypts while differentiated cells are localized to the villi. Indirect evidence has shown that the processes of differentiation and migration are driven in part by biochemical gradients of factors that specify the polarity of these cellular compartments; however, direct evidence for gradient-driven patterning of this in vivo architecture has been hampered by limitations of the in vitro systems available. Enteroid cultures are a powerful in vitro system; nevertheless, these spheroidal structures fail to replicate the architecture and lineage compartmentalization found in vivo, and are not easily subjected to gradients of growth factors. In the current work, we report the development of a micropatterned collagen scaffold with suitable extracellular matrix and stiffness to generate an in vitro self-renewing human small intestinal epithelium that replicates key features of the in vivo small intestine: a crypt-villus architecture with appropriate cell-lineage compartmentalization and an open and accessible luminal surface. Chemical gradients applied to the crypt-villus axis promoted the creation of a stem/progenitor-cell zone and supported cell migration along the crypt-villus axis. This new approach combining microengineered scaffolds, biophysical cues and chemical gradients to control the intestinal epithelium ex vivo can serve as a physiologically relevant mimic of the human small intestinal epithelium, and is broadly applicable to model other tissues that rely on gradients for physiological function.}, journal={BIOMATERIALS}, author={Wang, Yuli and Gunasekara, Duian B. and Reed, Mark I. and DiSalvo, Matthew and Bultman, Scott J. and Sims, Christopher E. and Magness, Scott T. and Allbritton, Nancy L.}, year={2017}, month={Jun}, pages={44–55} }
 @article{attayek_waugh_hunsucker_grayeski_sims_armistead_allbritton_2017, title={Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9}, volume={91}, ISSN={["1873-4235"]}, DOI={10.1016/j.bios.2016.12.019}, abstractNote={Microraft arrays have been used to screen and then isolate adherent and non-adherent cells with very high efficiency and excellent viability; however, manual screening and isolation limits the throughput and utility of the technology. In this work, novel hardware and software were developed to automate the microraft array platform. The developed analysis software identified microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity. The software enabled time-lapse imaging and the use of temporally varying characteristics as sort criteria. The automated hardware released microrafts with 98% efficiency and collected released microrafts with 100% efficiency. The automated system was used to examine the temporal variation in EGFP expression in cells transfected with CRISPR-Cas9 components for gene editing. Of 11,499 microrafts possessing a single cell, 220 microrafts were identified as possessing temporally varying EGFP-expression. Candidate cells (n=172) were released and collected from the microraft array and screened for the targeted gene mutation. Two cell colonies were successfully gene edited demonstrating the desired mutation.}, journal={BIOSENSORS & BIOELECTRONICS}, author={Attayek, Peter J. and Waugh, Jennifer P. and Hunsucker, Sally A. and Grayeski, Philip J. and Sims, Christopher E. and Armistead, Paul M. and Allbritton, Nancy L.}, year={2017}, month={May}, pages={175–182} }
 @article{proctor_sims_allbritton_2017, title={Chemical fixation to arrest phospholipid signaling for chemical cytometry}, volume={1523}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2017.05.022}, abstractNote={Chemical cytometry is a powerful tool for measuring biological processes such as enzymatic signaling at the single cell level. Among these technologies, single-cell capillary zone electrophoresis (CZE) has emerged as a powerful tool to assay a wide range of cellular metabolites. However, analysis of dynamic processes within cells remains challenging as signaling pathways are rapidly altered in response to changes in the cellular environment, including cell manipulation and storage. To address these limitations, we describe a method for chemical fixation of cells to stop the cellular reactions to preserve the integrity of key signaling molecules or reporters within the cell and to enable the cell to act as a storage reservoir for the reporter and its metabolites prior to assay by single-cell CZE. Fluorescent phosphatidylinositol 4,5-bisphosphate reporters were loaded into cells and the cells were chemically fixed and stored prior to analysis. The reporter and its metabolites were electrophoretically separated by single-cell CZE. Chemical fixation parameters such as fixative, fixation time, storage solution, storage duration, and extraction solution were optimized. When cells were loaded with a fluorescent C6- or C16-PIP2 followed by glutaraldehyde fixation and immediate analysis, 24±2% and 139±12% of the lipid was recoverable, respectively, when compared to an unfixed control. Storage of the cells for 24h yielded recoverable lipid of 61±3% (C6-PIP2) and 55±5% (C16-PIP2) when compared to cells analyzed immediately after fixation. The metabolites observed with and without fixation were identical. Measurement of phospholipase C activity in single leukemic cells in response to an agonist demonstrated the capability of chemical fixation coupled to single-cell CZE to yield an accurate snapshot of cellular reactions with the probe. This methodology enables cell assay with the reporter to be separated in space and time from reporter metabolite quantification while preserving assay integrity.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Proctor, Angela and Sims, Christopher E. and Allbritton, Nancy L.}, year={2017}, month={Nov}, pages={97–106} }
 @article{waybright_huang_proctor_wang_allbritton_zhang_2017, title={Required hydrophobicity of fluorescent reporters for phosphatidylinositol family of lipid enzymes}, volume={409}, ISSN={["1618-2650"]}, DOI={10.1007/s00216-017-0633-y}, abstractNote={The phosphatidylinositol (PtdIns) family of lipids plays important roles in cell differentiation, proliferation, and migration. Abnormal expression, mutation, or regulation of their metabolic enzymes has been associated with various human diseases such as cancer, diabetes, and bipolar disorder. Recently, fluorescent derivatives have increasingly been used as chemical probes to monitor either lipid localization or enzymatic activity. However, the requirements of a good probe have not been well defined, particularly modifications on the diacylglycerol side chain partly due to challenges in generating PtdIns lipids. We have synthesized a series of fluorescent PtdIns(4,5)P2 (PIP2) and PtdIns (PI) derivatives with various lengths of side chains and tested their capacity as substrates for PI3KIα and PI4KIIα, respectively. Both capillary electrophoresis and thin-layer chromatography were used to analyze enzymatic reactions. For both enzymes, the fluorescent probe with a longer side chain functions as a better substrate than that with a shorter chain and works well in the presence of the endogenous lipid, highlighting the importance of hydrophobicity of side chains in fluorescent phosphoinositide reporters. This comparison is consistent with their interactions with lipid vesicles, suggesting that the binding of a fluorescent lipid with liposome serves as a standard for assessing its utility as a chemical probe for the corresponding endogenous lipid. These findings are likely applicable to other lipid enzymes where the catalysis takes place at the lipid-water interface.}, number={29}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Waybright, Jarod and Huang, Weigang and Proctor, Angela and Wang, Xiaoyang and Allbritton, Nancy L. and Zhang, Qisheng}, year={2017}, month={Nov}, pages={6781–6789} }
 @article{wang_disalvo_gunasekara_dutton_proctor_lebhar_williamson_speer_howard_smiddy_et al._2017, title={Self-renewing monolayer of primary colonic or rectal epithelial cells}, volume={4}, number={1}, journal={Cellular and Molecular Gastroenterology and Hepatology}, author={Wang, Y. L. and DiSalvo, M. and Gunasekara, D. B. and Dutton, J. and Proctor, A. and Lebhar, M. S. and Williamson, I. A. and Speer, J. and Howard, R. L. and Smiddy, N. M. and et al.}, year={2017}, pages={165-} }
 @article{mainz_wang_lawrence_allbritton_2016, title={An Integrated Chemical Cytometry Method: Shining a Light on Akt Activity in Single Cells}, volume={55}, ISSN={["1521-3773"]}, DOI={10.1002/anie.201606914}, abstractNote={Abstract}, number={42}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Mainz, Emilie R. and Wang, Qunzhao and Lawrence, David S. and Allbritton, Nancy L.}, year={2016}, month={Oct}, pages={13095–13098} }
 @article{ornoff_wang_proctor_shah_allbritton_2016, title={Co-fabrication of chitosan and epoxy photoresist to form microwell arrays with permeable hydrogel bottoms}, volume={74}, ISSN={["1878-5905"]}, DOI={10.1016/j.biomaterials.2015.09.032}, abstractNote={Microfabrication technology offers the potential to create biological platforms with customizable patterns and surface chemistries, allowing precise control over the biochemical microenvironment to which a cell or group of cells is exposed. However, most microfabricated platforms grow cells on impermeable surfaces. This report describes the co-fabrication of a micropatterned epoxy photoresist film with a chitosan film to create a freestanding array of permeable, hydrogel-bottomed microwells. These films possess optical properties ideal for microscopy applications, and the chitosan layers are semi-permeable with a molecular exclusion of 9.9 ± 2.1 kDa. By seeding cells into the microwells, overlaying inert mineral oil, and supplying media via the bottom surface, this hybrid film permits cells to be physically isolated from one another but maintained in culture for at least 4 days. Arrays co-fabricated using these materials reduce both large-molecular-weight biochemical crosstalk between cells and mixing of different clonal populations, and will enable high-throughput studies of cellular heterogeneity with increased ability to customize dynamic interrogations compared to materials in currently available technologies.}, journal={BIOMATERIALS}, author={Ornoff, Douglas M. and Wang, Yuli and Proctor, Angela and Shah, Akash S. and Allbritton, Nancy L.}, year={2016}, month={Jan}, pages={77–88} }
 @article{proctor_zigoneanu_wang_sims_lawrence_allbritton_2016, title={Development of a protease-resistant reporter to quantify BCR-ABL activity in intact cells}, volume={141}, ISSN={["1364-5528"]}, DOI={10.1039/c6an01378c}, abstractNote={A peptidase-resistant ABL kinase substrate was developed by identifying protease-susceptible bonds on an ABL substrate peptide and replacing flanking amino acids with non-native amino acids.}, number={21}, journal={ANALYST}, author={Proctor, Angela and Zigoneanu, Imola G. and Wang, Qunzhao and Sims, Christopher E. and Lawrence, David S. and Allbritton, Nancy L.}, year={2016}, pages={6008–6017} }
 @article{attayek_hunsucker_sims_allbritton_armistead_2016, title={Identification and isolation of antigen-specific cytotoxic T lymphocytes with an automated microraft sorting system}, volume={8}, ISSN={["1757-9708"]}, DOI={10.1039/c6ib00168h}, abstractNote={The simultaneous measurement of T cell function with recovery of individual T cells would greatly facilitate characterizing antigen-specific responses bothin vivoand in model systems.}, number={12}, journal={INTEGRATIVE BIOLOGY}, author={Attayek, Peter J. and Hunsucker, Sally A. and Sims, Christopher E. and Allbritton, Nancy L. and Armistead, Paul M.}, year={2016}, pages={1208–1220} }
 @article{attayek_ahmad_wang_williamson_sims_magness_allbritton_2016, title={In vitro polarization of colonoids to create an intestinal stem cell compartment}, volume={11}, number={4}, journal={PLoS One}, author={Attayek, P. J. and Ahmad, A. A. and Wang, Y. L. and Williamson, I. and Sims, C. E. and Magness, S. T. and Allbritton, N. L.}, year={2016} }
 @article{turner_lebhae_proctor_wang_lawrence_allbritton_2016, title={Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity}, volume={11}, ISSN={["1554-8937"]}, DOI={10.1021/acschembio.5b00667}, abstractNote={Although peptide-based reporters of protein tyrosine kinase (PTK) activity have been used to study PTK enzymology in vitro, the application of these reporters to intracellular conditions is compromised by their dephosphorylation, preventing PTK activity measurements. Nonproteinogenic amino acids may be utilized to rationally design selective peptidic ligands by accessing greater chemical and structural diversity than is available using the native amino acids. We describe a peptidic reporter that, upon phosphorylation by the epidermal growth factor receptor (EGFR), is resistant to dephosphorylation both in vitro and in cellulo. The reporter contains a conformationally constrained phosphorylatable moiety (7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in the place of L-tyrosine and is efficiently phosphorylated in A431 epidermoid carcinoma cells. Dephosphorylation of the reporter occurs 3 orders of magnitude more slowly compared with that of the conventional tyrosine-containing reporter.}, number={2}, journal={ACS CHEMICAL BIOLOGY}, author={Turner, Abigail H. and Lebhae, Michael S. and Proctor, Angela and Wang, Qunzhao and Lawrence, David S. and Allbritton, Nancy L.}, year={2016}, month={Feb}, pages={355–362} }
 @article{mainz_serafin_nguyen_tarrant_sims_allbritton_2016, title={Single Cell Chemical Cytometry of Akt Activity in Rheumatoid Arthritis and Normal Fibroblast-like Synoviocytes in Response to Tumor Necrosis Factor alpha}, volume={88}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.6b01801}, abstractNote={The etiology of rheumatoid arthritis (RA) is poorly understood, and 30% of patients are unresponsive to established treatments targeting tumor necrosis factor α (TNFα). Akt kinase is implicated in TNFα signaling and may act as a barometer of patient responses to biologic therapies. Fluorescent peptide sensors and chemical cytometry were employed to directly measure Akt activity as well as proteolytic activity in individual fibroblast-like synoviocytes (FLS) from RA and normal subjects. The specificity of the peptide reporter was evaluated and shown to be a valid measure of Akt activity in single cells. The effect of TNFα treatment on Akt activity was highly heterogeneous between normal and RA subjects, which was not observable in bulk analyses. In 2 RA subjects, a bimodal distribution of Akt activity was observed, primarily due to a subpopulation (21.7%: RA Subject 5; 23.8%: RA Subject 6) of cells in which >60% of the reporter was phosphorylated. These subjects also possessed statistically elevated proteolytic cleavage of the reporter relative to normal subjects, suggesting heterogeneity in Akt and protease activity that may play a role in the RA-affected joint. We expect that chemical cytometry studies pairing peptide reporters with capillary electrophoresis will provide valuable data regarding aberrant kinase activity from small samples of clinical interest.}, number={15}, journal={ANALYTICAL CHEMISTRY}, author={Mainz, Emilie R. and Serafin, D. Stephen and Nguyen, Tuong T. and Tarrant, Teresa K. and Sims, Christopher E. and Allbritton, Nancy L.}, year={2016}, month={Aug}, pages={7786–7792} }
 @article{koh_hogue_wang_disalvo_allbritton_shi_olson_sosa_2016, title={Single-cell functional analysis of parathyroid adenomas reveals distinct classes of calcium sensing behaviour in primary hyperparathyroidism}, volume={20}, ISSN={["1582-4934"]}, DOI={10.1111/jcmm.12732}, abstractNote={Abstract}, number={2}, journal={JOURNAL OF CELLULAR AND MOLECULAR MEDICINE}, author={Koh, James and Hogue, Joyce A. and Wang, Yuli and DiSalvo, Matthew and Allbritton, Nancy L. and Shi, Yuhong and Olson, John A., Jr. and Sosa, Julie A.}, year={2016}, month={Feb}, pages={351–359} }
 @article{dickinson_meyer_pawlak_gomez_jaspers_allbritton_2015, title={Analysis of sphingosine kinase activity in single natural killer cells from peripheral blood}, volume={7}, ISSN={["1757-9708"]}, DOI={10.1039/c5ib00007f}, abstractNote={Heterogeneity of sphingosine kinase (SK) pathway activity in natural killer (NK) cells may enable cells to respond effectively to a diverse array of pathogens as well as incipient tumor cells.}, number={4}, journal={INTEGRATIVE BIOLOGY}, author={Dickinson, Alexandra J. and Meyer, Megan and Pawlak, Erica A. and Gomez, Shawn and Jaspers, Ilona and Allbritton, Nancy L.}, year={2015}, month={Apr}, pages={392–401} }
 @article{attayek_hunsucker_wang_sims_armistead_allbritton_2015, title={Array-Based Platform To Select, Release, and Capture Epstein-Barr Virus-Infected Cells Based on Intercellular Adhesion}, volume={87}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.5b03579}, abstractNote={Microraft arrays were developed to select and separate cells based on a complex phenotype, weak intercellular adhesion, without knowledge of cell-surface markers or intracellular proteins. Since the cells were also not competent to bind to a culture surface, a method to encapsulate nonadherent cells within a gelatin plug on the concave microraft surface was developed, enabling release and collection of the cells without the need for cell attachment to the microraft surface. After microraft collection, the gelatin was liquified to release the cell(s) for culture or analysis. A semiautomated release and collection device for the microrafts demonstrated 100 ± 0% collection efficiency of the microraft while increasing throughput 5-fold relative to that of manual release and collection. Using the microraft array platform along with the gelatin encapsulation method, single cells that were not surface-attached were isolated with a 100 ± 0% efficiency and a 96 ± 4% postsort single-cell cloning efficiency. As a demonstration, Epstein-Barr virus-infected lymphoblastoid cell lines (EBV-LCL) were isolated based on their intercellular adhesive properties. The identified cell colonies were collected with a 100 ± 0% sorting efficiency and a postsort viability of 87 ± 3%. When gene expression analysis of the EBV latency-associated gene, EBNA-2, was performed, there was no difference in expression between blasting or weakly adhesive cells and nonblasting or nonadhesive cells. Microraft arrays are a versatile method enabling separation of cells based on complicated and as yet poorly understood cell phenotypes.}, number={24}, journal={ANALYTICAL CHEMISTRY}, author={Attayek, Peter J. and Hunsucker, Sally A. and Wang, Yuli and Sims, Christopher E. and Armistead, Paul M. and Allbritton, Nancy L.}, year={2015}, month={Dec}, pages={12281–12289} }
 @article{gordon_wang_allbritton_taylor_2015, title={Magnetic Alignment of Microelements Containing Cultured Neuronal Networks for High-Throughput Screening}, volume={20}, ISSN={["1552-454X"]}, DOI={10.1177/1087057115598609}, abstractNote={High-throughput screening (HTS) on neurons presents unique difficulties because they are postmitotic, limited in supply, and challenging to harvest from animals or generate from stem cells. These limitations have hindered neurological drug discovery, leaving an unmet need to develop cost-effective technology for HTS using neurons. Traditional screening methods use up to 20,000 neurons per well in 384-well plates. To increase throughput, we use “microraft” arrays, consisting of 1600 square, releasable, paramagnetic, polystyrene microelements (microrafts), each providing a culture surface for 500–700 neurons. These microrafts can be detached from the array and transferred to 384-well plates for HTS; however, they must be centered within wells for automated imaging. Here, we developed a magnet array plate, compatible with HTS fluid-handling systems, to center microrafts within wells. We used finite element analysis to select an effective size of the magnets and confirmed that adjacent magnetic fields do not interfere. We then experimentally tested the plate’s centering ability and found a centering efficiency of 100%, compared with 4.35% using a flat magnet. We concluded that microrafts could be centered after settling randomly within the well, overcoming friction, and confirmed these results by centering microrafts containing hippocampal neurons cultured for 8 days.}, number={9}, journal={JOURNAL OF BIOMOLECULAR SCREENING}, author={Gordon, Kent R. and Wang, Yuli and Allbritton, Nancy L. and Taylor, Anne Marion}, year={2015}, month={Oct}, pages={1091–1100} }
 @article{ahmad_wang_sims_magness_allbritton_2015, title={Optimizing Wnt-3a and R-spondin1 concentrations for stem cell renewal and differentiation in intestinal organoids using a gradient-forming microdevice}, volume={5}, ISSN={["2046-2069"]}, DOI={10.1039/c5ra14923a}, abstractNote={A gradient-generating device assayed the impact of Wnt-3a and R-spondin1 on colonoids identifying concentrations required to yield a physiologically-relevant epithelium.}, number={91}, journal={RSC ADVANCES}, author={Ahmad, Asad A. and Wang, Yuli and Sims, Christopher E. and Magness, Scott T. and Allbritton, Nancy L.}, year={2015}, pages={74881–74891} }
 @article{mainz_dobes_allbritton_2015, title={Pronase E-Based Generation of Fluorescent Peptide Fragments: Tracking Intracellular Peptide Fate in Single Cells}, volume={87}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.5b01929}, abstractNote={The ability to track intracellular peptide proteolysis at the single cell level is of growing interest, particularly as short peptide sequences continue to play important roles as biosensors, therapeutics, and endogenous participants in antigen processing and intracellular signaling. We describe a rapid and inexpensive methodology to generate fluorescent peptide fragments from a parent sequence with diverse chemical properties, including aliphatic, nonpolar, basic, acidic, and non-native amino acids. Four peptide sequences with existing biochemical applications were fragmented using incubation with Pronase E and/or formic acid, and in each case a complete set of fluorescent fragments was generated for use as proteolysis standards in chemical cytometry. Fragment formation and identity was monitored with capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-MS) to confirm the presence of all sequences and yield fragmentation profiles across Pronase E concentrations which can readily be used by others. As a pilot study, Pronase E-generated standards from an Abl kinase sensor and an ovalbumin antigenic peptide were then employed to identify proteolysis products arising from the metabolism of these sequences in single cells. The Abl kinase sensor fragmented at 4.2 ± 4.8 zmol μM(-1) s(-1) and the majority of cells possessed similar fragment identities. In contrast, an ovalbumin epitope peptide was degraded at 8.9 ± 0.1 zmol μM(-1) s(-1), but with differential fragment formation between individual cells. Overall, Pronase E-generated peptide standards were a rapid and efficient method to identify proteolysis products from cells.}, number={15}, journal={ANALYTICAL CHEMISTRY}, author={Mainz, Emilie R. and Dobes, Nicholas C. and Allbritton, Nancy L.}, year={2015}, month={Aug}, pages={7987–7995} }
 @article{zigoneanu_sims_allbritton_2015, title={Separation of peptide fragments of a protein kinase C substrate fused to a beta-hairpin by capillary electrophoresis}, volume={407}, ISSN={["1618-2650"]}, DOI={10.1007/s00216-015-9065-8}, abstractNote={Synthetic peptides incorporating well-folded β-hairpin peptides possess advantages in a variety of cell biology applications by virtue of increased resistance to proteolytic degradation. In this study, the WKpG β-hairpin peptide fused to a protein kinase C (PKC) substrate was synthesized, and capillary-electrophoretic separation conditions for this peptide and its proteolytic fragments were developed. Fragments of WKpG-PKC were generated by enzymatic treatment with trypsin and Pronase E to produce standards for identification of degradation fragments in a cellular lysate. A simple buffer system of 250 mM H3PO4, pH 1.5 enabled separation of WKpG-PKC and its fragments by capillary electrophoresis in less than 16 min. Using a cellular lysate produced from Ba/F3 cells, the β-hairpin-conjugated substrate and its PKCα-phosphorylated product could be detected and separated from peptidase-generated fragments produced in a cell lysate. The method has potential application for identification and quantification of WKpG-PKC and its fragments in complex biological systems when the peptide is used as a reporter to assay PKC activity.}, number={30}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Zigoneanu, Imola G. and Sims, Christopher E. and Allbritton, Nancy L.}, year={2015}, month={Dec}, pages={8999–9008} }
 @article{grohman_gorelick_kottegoda_allbritton_rein_weeks_2014, title={An Immature Retroviral RNA Genome Resembles a Kinetically Trapped Intermediate State}, volume={88}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.03277-13}, abstractNote={ABSTRACT}, number={11}, journal={JOURNAL OF VIROLOGY}, author={Grohman, Jacob K. and Gorelick, Robert J. and Kottegoda, Sumith and Allbritton, Nancy L. and Rein, Alan and Weeks, Kevin M.}, year={2014}, month={Jun}, pages={6061–6068} }
 @article{shah_walker_sims_major_allbritton_2014, title={Dynamics and evolution of beta-catenin-dependent Wnt signaling revealed through massively parallel clonogenic screening}, volume={6}, ISSN={["1757-9708"]}, DOI={10.1039/c4ib00050a}, abstractNote={Wnt/β-catenin signaling is of significant interest due to the roles it plays in regulating development, tissue regeneration and disease. Transcriptional reporters have been widely employed to study Wnt/β-catenin signal transduction in live cells and whole organisms and have been applied to understanding embryonic development, exploring oncogenesis and developing therapeutics. Polyclonal heterogeneity in reporter cell lines has historically been seen as a challenge to be overcome in the development of novel cell lines and reporter-based assays, and monoclonal reporter cell lines are commonly employed to reduce this variability. A375 cell lines infected with a reporter for Wnt/β-catenin signaling were screened over short (<6) and long (>25) generational timescales. To characterize phenotypic divergence over these time-scales, a microfabricated cell array-based screen was developed enabling characterization of 1119 clonal colonies in parallel. This screen revealed phenotypic divergence after <6 generations at a similar scale to that observed in monoclonal cell lines cultured for >25 generations. Not only were reporter dynamics observed to diverge widely, but monoclonal cell lines were observed with seemingly opposite signaling phenotypes. Additionally, these observations revealed a generational-dependent trend in Wnt signaling in A375 cells that provides insight into the pathway's mechanisms of positive feedback and self-inhibition.}, number={7}, journal={INTEGRATIVE BIOLOGY}, author={Shah, Pavak K. and Walker, Matthew P. and Sims, Christopher E. and Major, Michael B. and Allbritton, Nancy L.}, year={2014}, pages={673–684} }
 @article{phillips_dailey_bair_samet_allbritton_2014, title={Ex Vivo Chemical Cytometric Analysis of Protein Tyrosine Phosphatase Activity in Single Human Airway Epithelial Cells}, volume={86}, ISSN={["1520-6882"]}, DOI={10.1021/ac403705c}, abstractNote={We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min(-1) mg(-1)) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (76%, 69%, and 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min(-1) mg(-1) and was also effectively inhibited by preincubation of the cells with the inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min(-1) mg(-1) resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34 to 36 pmol min(-1) mg(-1) (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues.}, number={2}, journal={ANALYTICAL CHEMISTRY}, author={Phillips, Ryan M. and Dailey, Lisa A. and Bair, Eric and Samet, James M. and Allbritton, Nancy L.}, year={2014}, month={Jan}, pages={1291–1297} }
 @article{proctor_herrera-loeza_wang_lawrence_yeh_allbritton_2014, title={Measurement of Protein Kinase B Activity in Single Primary Human Pancreatic Cancer Cells}, volume={86}, ISSN={["1520-6882"]}, DOI={10.1021/ac500616q}, abstractNote={An optimized peptide substrate was used to measure protein kinase B (PKB) activity in single cells. The peptide substrate was introduced into single cells, and capillary electrophoresis was used to separate and quantify nonphosphorylated and phosphorylated peptide. The system was validated in three model pancreatic cancer cell lines before being applied to primary cells from human pancreatic adenocarcinomas propagated in nude mice. As measured by phosphorylation of peptide substrate, each tumor cell line exhibited statistically different median levels of PKB activity (65%, 21%, and 4% phosphorylation in PANC-1 (human pancreatic carcinoma), CFPAC-1 (human metastatic ductal pancreatic adenocarcinoma), and HPAF-II cells (human pancreatic adenocarcinoma), respectively) with CFPAC-1 cells demonstrating two populations of cells or bimodal behavior in PKB activation levels. The primary cells exhibited highly variable PKB activity at the single cell level, with some cells displaying little to no activity and others possessing very high levels of activity. This system also enabled simultaneous characterization of peptidase action in single cells by measuring the amount of cleaved peptide substrate in each cell. The tumor cell lines displayed degradation rates statistically similar to one another (0.02, 0.06, and 0.1 zmol pg–1 s–1, for PANC-1, CFPAC-1, and HPAF-II cells, respectively) while the degradation rate in primary cells was 10-fold slower. The peptide cleavage sites also varied between tissue-cultured and primary cells, with 5- and 8-residue fragments formed in tumor cell lines and only the 8-residue fragment formed in primary cells. These results demonstrate the ability of chemical cytometry to identify important differences in enzymatic behavior between primary cells and tissue-cultured cell lines.}, number={9}, journal={ANALYTICAL CHEMISTRY}, author={Proctor, Angela and Herrera-Loeza, S. Gabriela and Wang, Qunzhao and Lawrence, David S. and Yeh, Jen Jen and Allbritton, Nancy L.}, year={2014}, month={May}, pages={4573–4580} }
 @article{gach_attayek_whittlesey_yeh_allbritton_2014, title={Micropallet arrays for the capture, isolation and culture of circulating tumor cells from whole blood of mice engrafted with primary human pancreatic adenocarcinoma}, volume={54}, ISSN={["1873-4235"]}, DOI={10.1016/j.bios.2013.11.019}, abstractNote={Circulating tumor cells (CTCs) are important biomarkers of cancer progression and metastatic potential. The rarity of CTCs in peripheral blood has driven the development of technologies to isolate these tumor cells with high specificity; however, there are limited techniques available for isolating target CTCs following enumeration. A strategy is described to capture and isolate viable tumor cells from whole blood using an array of releasable microstructures termed micropallets. Specific capture of nucleated cells or cells expressing epithelial cell adhesion molecules (EpCAM) was achieved by functionalizing micropallet surfaces with either fibronectin, Matrigel or anti-EpCAM antibody. Surface grafting of poly(acrylic acid) followed by covalent binding of protein A/G enabled efficient capture of EpCAM antibody on the micropallet surface. MCF-7 cells, a human breast adenocarcinoma, were retained on the array surface with 90±8% efficiency when using an anti-EpCAM-coated array. To demonstrate the efficiency of tumor cell retention on micropallet arrays in the presence of blood, MCF-7 cells were mixed into whole blood and added to small arrays (71 mm2) coated with fibronectin, Matrigel or anti-EpCAM. These approaches achieved MCF-7 cell capture from ≤10 µL of whole blood with efficiencies greater than 85%. Furthermore, MCF-7 cells intermixed with 1 mL blood and loaded onto large arrays (7171 mm2) were captured with high efficiencies (≥97%), could be isolated from the array by a laser-based approach and were demonstrated to yield a high rate of colony formation (≥85%) after removal from the array. Clinical utility of this technology was shown through the capture, isolation and successful culture of CTCs from the blood of mice engrafted with primary human pancreatic tumors. Direct capture and isolation of living tumor cells from blood followed by analysis or culture will be a valuable tool for cancer cell characterization.}, journal={BIOSENSORS & BIOELECTRONICS}, author={Gach, Philip C. and Attayek, Peter J. and Whittlesey, Rebecca L. and Yeh, Jen Jen and Allbritton, Nancy L.}, year={2014}, month={Apr}, pages={476–483} }
 @article{kovarik_dickinson_roy_poonnen_fine_allbritton_2014, title={Response of single leukemic cells to peptidase inhibitor therapy across time and dose using a microfluidic device}, volume={6}, ISSN={["1757-9708"]}, DOI={10.1039/c3ib40249e}, abstractNote={Microfluidic single-cell assays of peptide degradation were performed at varying inhibitor doses, and the resulting data were analyzed by regression modeling to reveal biological effects.}, number={2}, journal={INTEGRATIVE BIOLOGY}, author={Kovarik, Michelle L. and Dickinson, Alexandra J. and Roy, Pourab and Poonnen, Ranjit A. and Fine, Jason P. and Allbritton, Nancy L.}, year={2014}, pages={164–174} }
 @article{dickinson_hunsucker_armistead_allbritton_2014, title={Single-cell sphingosine kinase activity measurements in primary leukemia}, volume={406}, ISSN={["1618-2650"]}, DOI={10.1007/s00216-014-7974-6}, abstractNote={Sphingosine kinase (SK) is a promising therapeutic target in a number of cancers, including leukemia. Traditionally, SK has been measured in bulk cell lysates, but this technique obscures the cellular heterogeneity present in this pathway. For this reason, SK activity was measured in single cells loaded with a fluorescent sphingosine reporter. An automated capillary electrophoresis (CE) system enabled rapid separation and quantification of the phosphorylated and nonphosphorylated sphingosine reporter in single cells. SK activity was measured in tissue-cultured cells derived from chronic myelogenous leukemia (K562), primary peripheral blood mononuclear cells (PBMCs) from three patients with different forms of leukemia, and enriched leukemic blasts from a patient with acute myeloid leukemia (AML). Significant intercellular heterogeneity existed in terms of the degree of reporter phosphorylation (as much as an order of magnitude difference), the amount of reporter uptake, and the metabolites formed. In K562 cells, the average amount of reporter converted to the phosphorylated form was 39 ± 26 % per cell. Of the primary PBMCs analyzed, the average amount of phosphorylated reporter was 16 ± 25 %, 11 ± 26 %, and 13 ± 23 % in a chronic myelogenous leukemia (CML) patient, an AML patient, and a B-cell acute lymphocytic leukemia (B-ALL) patient, respectively. These experiments demonstrated the challenge of studying samples comprised of multiple cell types, with tumor blasts present at 5 to 87 % of the cell population. When the leukemic blasts from a fourth patient with AML were enriched to 99 % of the cell population, 19 ± 36 % of the loaded sphingosine was phosphorylated. Thus, the diversity in SK activity remained even in a nearly pure tumor sample. These enriched AML blasts loaded significantly less reporter (0.12 ± 0.2 amol) relative to that loaded into the PBMCs in the other samples (≥1 amol). The variability in SK signaling may have important implications for SK inhibitors as therapeutics for leukemia and demonstrates the value of single-cell analysis in characterizing the nature of oncogenic signaling in cancer.}, number={27}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Dickinson, Alexandra J. and Hunsucker, Sally A. and Armistead, Paul M. and Allbritton, Nancy L.}, year={2014}, month={Nov}, pages={7027–7036} }
 @article{shah_herrera-loeza_sims_yeh_allbritton_2014, title={Small Sample Sorting of Primary Adherent Cells by Automated Micropallet Imaging and Release}, volume={85A}, ISSN={["1552-4930"]}, DOI={10.1002/cyto.a.22480}, abstractNote={Abstract}, number={7}, journal={CYTOMETRY PART A}, author={Shah, Pavak K. and Herrera-Loeza, Silvia Gabriela and Sims, Christopher E. and Yeh, Jen Jen and Allbritton, Nancy L.}, year={2014}, month={Jul}, pages={642–649} }
 @article{melvin_woss_park_dumberger_waters_allbritton_2013, title={A comparative analysis of the ubiquitination kinetics of multiple degrons to identify an ideal targeting sequence for a proteasome reporter}, volume={8}, number={10}, journal={PLoS One}, author={Melvin, A. T. and Woss, G. S. and Park, J. H. and Dumberger, L. D. and Waters, M. L. and Allbritton, N. L.}, year={2013} }
 @article{wang_phillips_herrera_sims_yeh_allbritton_2013, title={Array of biodegradable microrafts for isolation and implantation of living, adherent cells}, volume={3}, number={24}, journal={RSC Advances}, author={Wang, Y. L. and Phillips, C. N. and Herrera, G. S. and Sims, C. E. and Yeh, J. J. and Allbritton, N. L.}, year={2013}, pages={9264–9272} }
 @article{dickinson_armistead_allbritton_2013, title={Automated Capillary Electrophoresis System for Fast Single-Cell Analysis}, volume={85}, ISSN={["1520-6882"]}, DOI={10.1021/ac4005887}, abstractNote={Capillary electrophoresis (CE) is a promising technique for single-cell analysis, but its use in biological studies has been limited by low throughput. This paper presents an automated platform employing microfabricated cell traps and a three-channel system for rapid buffer exchange for fast single-cell CE. Cells loaded with fluorescein and Oregon green were analyzed at a throughput of 3.5 cells/min with a resolution of 2.3 ± 0.6 for the fluorescein and Oregon green. Cellular protein kinase B (PKB) activity, as measured by immunofluorescence staining of phospho-PKB, was not altered, suggesting that this stress-activated kinase was not upregulated during the CE experiments and that basal cell physiology was not perturbed prior to cell lysis. The activity of sphingosine kinase (SK), which is often upregulated in cancer, was measured in leukemic cells by loading a sphingosine-fluorescein substrate into cells. Sphingosine fluorescein (SF), sphingosine-1-phosphate fluorescein (S1PF), and a third fluorescent species were identified in single cells. A single-cell throughput of 2.1 cells/min was achieved for 219 total cells. Eighty-eight percent of cells possessed upregulated SK activity, although subpopulations of cells with markedly different SK activity relative to that of the population average were readily identified. This system was capable of stable and reproducible separations of biological compounds in hundreds of adherent and nonadherent cells, enabling measurements of previously uncharacterized biological phenomena.}, number={9}, journal={ANALYTICAL CHEMISTRY}, author={Dickinson, Alexandra J. and Armistead, Paul M. and Allbritton, Nancy L.}, year={2013}, month={May}, pages={4797–4804} }
 @article{wang_ahmad_shah_sims_magness_allbritton_2013, title={Capture and 3D culture of colonic crypts and colonoids in a microarray platform}, volume={13}, number={23}, journal={Lab on a Chip}, author={Wang, Y. and Ahmad, A. A. and Shah, P. K. and Sims, C. E. and Magness, S. T. and Allbritton, N. L.}, year={2013}, pages={4625–4634} }
 @article{ornoff_wang_allbritton_2013, title={Characterization of freestanding photoresist films for biological and MEMS applications}, volume={23}, number={2}, journal={Journal of Micromechanics and Microengineering}, author={Ornoff, D. M. and Wang, Y. and Allbritton, N. L.}, year={2013} }
 @article{pai_kluckman_cowley_bortner_sims_allbritton_2013, title={Efficient division and sampling of cell colonies using microcup arrays}, volume={138}, ISSN={["1364-5528"]}, DOI={10.1039/c2an36065a}, abstractNote={A microengineered array to sample clonal colonies is described. The cells were cultured on an array of individually releasable elements until the colonies expanded to cover multiple elements. Single elements were released using a laser-based system and collected to sample cells from individual colonies. A greater than an 85% rate in splitting and collecting colonies was achieved using a 3-dimensional cup-like design or "microcup". Surface modification using patterned titanium deposition of the glass substrate improved the stability of microcup adhesion to the glass while enabling minimization of the laser energy for splitting the colonies. Smaller microcup dimensions and slotting the microcup walls reduced the time needed for colonies to expand into multiple microcups. The stem cell colony retained on the array and the collected fraction within released microcups remained undifferentiated and viable. The colony samples were characterized by both reporter gene expression and a destructive assay (PCR) to identify target colonies. The platform is envisioned as a means to rapidly establish cell lines using a destructive assay to identify desired clones.}, number={1}, journal={ANALYST}, author={Pai, Jeng-Hao and Kluckman, Kimberly and Cowley, Dale O. and Bortner, Donna M. and Sims, Christopher E. and Allbritton, Nancy L.}, year={2013}, pages={220–228} }
 @article{balowski_wang_allbritton_2013, title={Fabrication of 3D Microstructures from Interactions of Immiscible Liquids with a Structured Surface}, volume={25}, ISSN={["1521-4095"]}, DOI={10.1002/adma.201301658}, abstractNote={A new lithography technique is presented that exploits the interactions of immiscible liquids with a structured surface. This highly parallel, "low-tech" method requires no dedicated equipment and easily produces curved and/or multi-level structures out of a variety of photoactive and non-photoactive materials.}, number={30}, journal={ADVANCED MATERIALS}, author={Balowski, Joseph J. and Wang, Yuli and Allbritton, Nancy L.}, year={2013}, month={Aug}, pages={4107–4112} }
 @article{gach_attayek_herrera_yeh_allbritton_2013, title={Isolation and in Vitro Culture of Rare Cancer Stem Cells from Patient-Derived Xenografts of Pancreatic Ductal Adenocarcinoma}, volume={85}, ISSN={["1520-6882"]}, DOI={10.1021/ac401165s}, abstractNote={Described is the construction of a large array of releasable microstructures (micropallets) along with screening and isolation protocols for sorting rare, approximately 1 in 10,000, cancer stem cells (CSCs) from a heterogeneous cell population. A 10.1 × 7.1 cm array of micropallets (50 × 50 × 75 μm structures and 25 μm micropallet gap) was fabricated on a large glass substrate, providing an array of approximately 1.3 million releasable microstructures. Image analysis algorithms were developed to permit array screening for identification of fluorescently labeled cells in less than 15 min using an epifluorescent wide-field microscope with a computer controlled translational stage. Device operation was tested by culturing HeLa cells transfected with green fluorescent protein (GFP) admixed with wild-type HeLa cells at ratios of 1:10(4) to 1:10(6) on the array followed by screening to identify flourescent cells. Micropallets containing cells of interest were then selectively released by a focused laser pulse and collected on a numbered poly(dimethylsiloxane) (PDMS) substrate with high viability. A direct comparison of this technology with fluorescence-activated cell sorting (FACS) demonstrated that micropallet arrays offered enhanced post sorting purity (100%), yield (100%), and viability (94-100%) for rare cell isolation. As a demonstration of the technology's value, pancreatic tumor cells from Panc-1 cell lines and patient-derived xenografts were screened for the presence of CD24, CD44, and CD326: surface markers of pancreatic CSCs. Following cell isolation and culture, 63 ± 23% of the isolated Panc-1 cells and 35% of sorted human xenograft cells formed tumor spheroids retaining high expression levels of CD24, CD44, and CD326. The ability to isolate rare cells from relatively small sample sizes will facilitate our understanding of cell biology and the development of new therapeutic strategies.}, number={15}, journal={ANALYTICAL CHEMISTRY}, author={Gach, Philip C. and Attayek, Peter J. and Herrera, Gabriela and Yeh, Jen Jen and Allbritton, Nancy L.}, year={2013}, month={Aug}, pages={7271–7278} }
 @article{dobes_dhopeshwarkar_henley_ramsey_sims_allbritton_2013, title={Laser-based directed release of array elements for efficient collection into targeted microwells}, volume={138}, ISSN={["1364-5528"]}, DOI={10.1039/c2an36342a}, abstractNote={A cell separation strategy capable of the systematic isolation and collection of moderate to large numbers (25-400) of single cells into a targeted microwell is demonstrated. An array of microfabricated, releasable, transparent micron-scale pedestals termed pallets and an array of microwells in poly(dimethylsiloxane) (PDMS) were mated to enable selective release and retrieval of individual cells. Cells cultured on a pallet array mounted on a custom designed stage permitted the array to be positioned independently of the microwell locations. Individual pallets containing cells were detached in a targeted fashion using a pulsed Nd:YAG laser. The location of the laser focal point was optimized to transfer individual pallets to designated microwells. In a large-scale sort (n = 401), the accuracy, defined as placing a pallet in the intended well, was 94% and the collection efficiency was 100%. Multiple pallets were observed in only 4% of the targeted wells. In cell sorting experiments, the technique provided a yield and purity of target cells identified by their fluorescence signature of 91% and 93%, respectively. Cell viability based on single-cell cloning efficiency at 72 h post collection was 77%.}, number={3}, journal={ANALYST}, author={Dobes, Nicholas C. and Dhopeshwarkar, Rahul and Henley, W. Hampton and Ramsey, J. Michael and Sims, Christopher E. and Allbritton, Nancy L.}, year={2013}, pages={831–838} }
 @article{phillips_bair_lawrence_sims_allbritton_2013, title={Measurement of Protein Tyrosine Phosphatase Activity in Single Cells by Capillary Electrophoresis}, volume={85}, ISSN={["1520-6882"]}, DOI={10.1021/ac401106e}, abstractNote={A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn(2+) and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min(-1) mg(-1)) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min(-1) mg(-1)). Zn(2+), 1,2-naphthoquinone, and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 μM, and 100 nM, respectively. Dephosphorylation of the reporter, following loading into living single cells, occurred at rates of at least 2 pmol min(-1) mg(-1). When single cells were exposed to 1,2-naphthoquinone (50 μM), Zn(2+) (100 μM), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens.}, number={12}, journal={ANALYTICAL CHEMISTRY}, author={Phillips, Ryan M. and Bair, Eric and Lawrence, David S. and Sims, Christopher E. and Allbritton, Nancy L.}, year={2013}, month={Jun}, pages={6136–6142} }
 @article{melvin_woss_park_waters_allbritton_2013, title={Measuring Activity in the Ubiquitin-Proteasome System: From Large Scale Discoveries to Single Cells Analysis}, volume={67}, ISSN={["1085-9195"]}, DOI={10.1007/s12013-013-9621-9}, abstractNote={The ubiquitin–proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS has provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington’s disease. These reporters, usually consisting of a recognition sequence fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes. This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, a recent study is presented highlighting the development of a novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples.}, number={1}, journal={CELL BIOCHEMISTRY AND BIOPHYSICS}, author={Melvin, Adam T. and Woss, Gregery S. and Park, Jessica H. and Waters, Marcey L. and Allbritton, Nancy L.}, year={2013}, month={Sep}, pages={75–89} }
 @misc{kovarik_ornoff_melvin_dobes_wang_dickinson_gach_shah_allbritton_2013, title={Micro Total Analysis Systems: Fundamental Advances and Applications in the Laboratory, Clinic, and Field}, volume={85}, ISSN={["1520-6882"]}, DOI={10.1021/ac3031543}, abstractNote={Applications of micro total analysis systems (μTAS) span basic-science research, clinical medicine, and field work. Assay devices designed for these applications offer improvements to existing methods or provide fundamentally new strategies. Both mature methods and novel techniques have benefited from the increased throughput, integration and miniaturization afforded by μTAS. Traditional assays such Western blots and binding assays are recapitulated in a μTAS format but with reduced reagent usage, decreased performance times and added capabilities. An increasingly vibrant area is the performance of drug screening and toxicology assays on-chip, enabling the efficient screening of very large numbers of molecules. Similarly, recent μTAS reactors demonstrate greater chemical synthetic yields and novel product synthesis compared to macro-systems, often as a result of accurate control over reaction conditions including precision reagent dispensing. These exciting systems are now enabling on-site production of short-lived radioactive compounds for medical applications. The greatest impact of μTAS may very well be the ability to perform massively parallel laboratory experiments, for example, the use millions of reaction vessels or the analysis of hundreds of thousands of single cells. Another strength of μTAS lies in the creation of multicellular communities, for example, the combination of many cell types into an interacting system to explore intercellular communication. Devices with multiple layers of co-cultured tissues benefit from precise placement of molecules, such as extracellular matrices or growth factors, in both space and time. Similarly, the complexity and variety of organ-on-chip and organism-on-chip technologies continues to escalate rapidly. Impressively, the types of organisms cultured on-chip now range from the simplest bacteria to complex animals such as fish. 
 
Automation, reliability, and integration must all increase as a device moves from the specialist environment of a lab to usage by non-expert personnel in the outside world, for example, at a clinical point-of-care or in environmental monitoring. Key innovations in recent months result in devices that operate with minimal external equipment, error-free operation, and unambiguous readouts, all critical for operation by untrained personnel. Lightweight, portable devices are increasingly used to identify chemical and biological toxins in water, air and soil with applications in public health, defense, and homeland security. Perhaps most exciting is the development of μTAS with sufficient robustness for operation in challenging environments, such as the ocean and outer space. A central component of these systems is the ability to withstand the unexpected. These systems push the boundaries of current integration principles and spur rapid growth of new design philosophies. 
 
This review focuses on advances in the area of μTAS or “lab-on-a-chip” systems over the time span of May 2011 through September 2012 with a focus on applications in basic research, clinical medicine and field usage. A range of journals with 2011 impact factors from 2.0 to 36.3 were screened to cover publications with highly specialized content as well as those directed at multidisciplinary audiences. These publications included discipline-specific journals such as Analytical Chemistry and Lab on a Chip as well as general scientific publications, e.g. Science and Nature. To identify material beyond the individually examined journals, extensive key word searches in databases such as PubMed, SciFinder, and Web of Science were performed. Recent reviews in the area of μTAS were also examined for appropriate references. Care was taken to identify impactful and exciting work from across the globe. Well over a thousand papers in the three target areas were identified and discussed. Due to space limitations, we were unable to include all papers but instead incorporated those most fitting into the review scheme and those reporting innovations in basic microdevice technology as well as in applications to biological, physical and engineering sciences. We apologize in advance for omitted papers and welcome feedback regarding any oversights on our part.}, number={2}, journal={ANALYTICAL CHEMISTRY}, author={Kovarik, Michelle L. and Ornoff, Douglas M. and Melvin, Adam T. and Dobes, Nicholas C. and Wang, Yuli and Dickinson, Alexandra J. and Gach, Philip C. and Shah, Pavak K. and Allbritton, Nancy L.}, year={2013}, month={Jan}, pages={451–472} }
 @article{kovarik_shah_armistead_allbritton_2013, title={Microfluidic Chemical Cytometry of Peptide Degradation in Single Drug-Treated Acute Myeloid Leukemia Cells}, volume={85}, ISSN={["1520-6882"]}, DOI={10.1021/ac4002029}, abstractNote={Microfluidic systems show great promise for single-cell analysis; however, as these technologies mature, their utility must be validated by studies of biologically relevant processes. An important biomedical application of these systems is characterization of tumor cell heterogeneity. In this work, we used a robust microfluidic platform to explore the heterogeneity of enzyme activity in single cells treated with a chemotherapeutic drug. Using chemical cytometry, we measured peptide degradation in the U937 acute myeloid leukemia (AML) cell line in the presence and absence of the aminopeptidase inhibitor Tosedostat (CHR-2797). The analysis of 99 untreated cells revealed rapid and consistent degradation of the peptide reporter within 20 min of loading. Results from drug-treated cells showed inhibited, but ongoing degradation of the reporter. Because the device operates at an average sustained throughput of 37 ± 7 cells/h, we were able to sample cells over the course of this time-dependent degradation. In data from 498 individual drug-treated cells, we found a linear dependence of degradation rate on amount of substrate loaded superimposed upon substantial heterogeneity in peptide processing in response to inhibitor treatment. Importantly, these data demonstrated the potential of microfluidic systems to sample biologically relevant analytes and time-dependent processes in large numbers of single cells.}, number={10}, journal={ANALYTICAL CHEMISTRY}, author={Kovarik, Michelle L. and Shah, Pavak K. and Armistead, Paul M. and Allbritton, Nancy L.}, year={2013}, month={May}, pages={4991–4997} }
 @article{shah_hughes_wang_sims_allbritton_2013, title={Scalable synthesis of a biocompatible, transparent and superparamagnetic photoresist for microdevice fabrication}, volume={23}, number={10}, journal={Journal of Micromechanics and Microengineering}, author={Shah, P. K. and Hughes, M. R. and Wang, Y. and Sims, C. E. and Allbritton, N. L.}, year={2013} }
 @article{yang_proctor_cline_houston_waters_allbritton_2013, title={beta-Turn sequences promote stability of peptide substrates for kinases within the cytosolic environment}, volume={138}, number={15}, journal={Analyst [London]}, author={Yang, S. and Proctor, A. and Cline, L. L. and Houston, K. M. and Waters, M. L. and Allbritton, N. L.}, year={2013}, pages={4305–4311} }
 @article{proctor_wang_lawrence_allbritton_2012, title={Development of a Peptidase-Resistant Substrate for Single-Cell Measurement of Protein Kinase B Activation}, volume={84}, ISSN={["1520-6882"]}, DOI={10.1021/ac301489d}, abstractNote={An iterative design strategy using three criteria was utilized to develop a peptidase-resistant substrate peptide for protein kinase B. Libraries of peptides possessing non-native amino acids were screened for time to 50% phosphorylation, degradation half-life within a lysate, and appearance of a dominant fragment. The lead peptide possessed a half-life of 92 ± 7 and 16 ± 2 min in HeLa and LNCaP cytosolic lysates, respectively, representing a 4.6- and 2.7-fold lifetime improvement over that of the starting peptide. The redesigned peptide possessed a 4.5-fold improvement in phosphorylation efficiency compared to the starting peptide. The same peptide fragments were formed when the lead peptide was incubated in a lysate or loaded into single cells although the fragments formed in significantly different ratios suggesting that distinct peptidases metabolized the peptide in the two preparations. The rate of peptide degradation and phosphorylation was on average 0.1 ± 0.2 zmol pg(-1) s(-1) and 0.04 ± 0.08 zmol pg(-1) s(-1), respectively, for single LNCaP cells loaded with 4 ± 8 μM of peptide. Peptidase-resistant kinase substrates should find widespread utility in both lysate-based and single-cell assays of kinase activity.}, number={16}, journal={ANALYTICAL CHEMISTRY}, author={Proctor, Angela and Wang, Qunzhao and Lawrence, David S. and Allbritton, Nancy L.}, year={2012}, month={Aug}, pages={7195–7202} }
 @article{wang_sims_allbritton_2012, title={Dissolution-guided wetting for microarray and microfluidic devices}, volume={12}, number={17}, journal={Lab on a Chip}, author={Wang, Y. L. and Sims, C. E. and Allbritton, N. L.}, year={2012}, pages={3036–3039} }
 @article{proctor_wang_lawrence_allbritton_2012, title={Metabolism of peptide reporters in cell lysates and single cells}, volume={137}, number={13}, journal={Analyst [London]}, author={Proctor, A. and Wang, Q. Z. and Lawrence, D. S. and Allbritton, N. L.}, year={2012}, pages={3028–3038} }
 @misc{kovarik_gach_ornoff_wang_balowski_farrag_allbritton_2012, title={Micro Total Analysis Systems for Cell Biology and Biochemical Assays}, volume={84}, ISSN={["1520-6882"]}, DOI={10.1021/ac202611x}, abstractNote={Novel applications of micro total analysis systems (µTAS) are addressing fundamental biological questions, creating new biomedical reagents, and developing innovative cell and biochemical assays. These efforts impact progress in all areas of µTAS from materials to fluidic handling as well as detection and external control systems. Three areas show the greatest current and potential impact on the biomedical sciences: improvements in device fabrication and operation, development of enabling technologies, and advancements at the interface with biology (Figure 1). The range of materials from which devices can be fabricated has expanded considerably and now includes paper, fabric and thread, and a multitude of polymers as well as more conventional materials. Thus device substrates and component materials suitable for nearly all biological applications are readily available. Devices are also becoming increasingly integrated with advancements in sample handling and preparation, key first steps in any biological analysis. Another growing area focuses on modular components that can be mixed and matched on-demand and applied to many different assays, so-called programmable microfluidics. This development should enhance the rate at which new bioassays are generated as well as customize existing experimental protocols. A second area of rapid advancement has been the development new technologies that enable assays that cannot be efficiently performed by any method except µTAS. Novel analyses of single cells are enabled due to effective manipulation of picoliter-scale volumes. Synthesis and screening of large-scale libraries has become increasingly feasible due to the fast processing speeds and combinatorial mixing of reagents provided by lab-on-chip systems. Increased automation within a completely contained system has now begun to provide some of the first true µTAS diagnostic devices for clinical medicine. The third area in which µTAS has begun to yield high dividends is the interfacing of living entities with microdevices to create biological communities, including tissues and organs on-chip. Control of cell placement in multiple dimensions has produced biological systems midway between the conventional tissue-culture dish and an intact animal. Thus the complexities of living constructs can be recreated in a controlled experimental environment permitting groundbreaking biological questions to be addressed. Application of µTAS in all of these areas continues to be highly interdisciplinary, utilizing techniques and strategies from almost every scientific field. This multidisciplinary focus insures continued relevance to the biological community as well as a bright future. 
 
 
 
Figure 1 
 
We highlight recent contributions to µTAS in three interlocking areas: fabrication & operation, enabling technologies, and interfacing with biology. 
 
 
 
Due to the rapid progress of µTAS or “lab-on-a-chip” systems, this review focuses on advances impacting cell biology and biochemistry and covers the time span from March 2010 through August 2011. The material for the review was compiled using several strategies: reviews of high impact journals such as Analytical Chemistry, Lab on a Chip, Science, Nature, and PNAS; extensive key word searches in databases such as PubMed, SciFinder, Web of Science, and Google Scholar; and screens of other recent topical reviews. Although several thousand papers were identified and over a thousand papers received a detailed examination, we focused on the most novel and exciting methods, devices, and applications in the areas of cell biology and biochemistry. We also endeavored to cover the most prominent work from a range of labs and countries. Ultimately we were limited by space constraints and our desire to craft a readable commentary on the state of the field. We apologize in advance for omitted papers and welcome feedback regarding any oversights on our part.}, number={2}, journal={ANALYTICAL CHEMISTRY}, author={Kovarik, Michelle L. and Gach, Philip C. and Ornoff, Douglas M. and Wang, Yuli and Balowski, Joseph and Farrag, Lila and Allbritton, Nancy L.}, year={2012}, month={Jan}, pages={516–540} }
 @article{gach_xu_king_sims_bear_allbritton_2012, title={Microfabricated Arrays for Splitting and Assay of Clonal Colonies}, volume={84}, ISSN={["1520-6882"]}, DOI={10.1021/ac301895t}, abstractNote={A microfabricated platform was developed for highly parallel and efficient colony picking, splitting, and clone identification. A pallet array provided patterned cell colonies which mated to a second printing array composed of bridging microstructures formed by a supporting base and attached post. The posts enabled mammalian cells from colonies initially cultured on the pallet array to migrate to corresponding sites on the printing array. Separation of the arrays simultaneously split the colonies, creating a patterned replica. Optimization of array elements provided transfer efficiencies greater than 90% using bridging posts of 30 μm diameter and 100 μm length and total colony numbers of 3000. Studies using five mammalian cell lines demonstrated that a variety of adherent cell types could be cultured and effectively split with printing efficiencies of 78-92%. To demonstrate the technique's utility, clonal cell lines with siRNA knockdown of Coronin 1B were generated using the arrays and compared to a traditional FACS/Western Blotting-based approach. Identification of target clones required a destructive assay to identify cells with an absence of Coronin 1B brought about by the successful infection of interfering shRNA construct. By virtue of miniaturization and its parallel format, the platform enabled the identification and generation of 12 target clones from a starting sample of only 3900 cells and required only 5 man hours over 11 days. In contrast, the traditional method required 500,000 cells and generated only 5 target clones with 34 man hours expended over 47 days. These data support the considerable reduction in time, manpower, and reagents using the miniaturized platform for clonal selection by destructive assay versus conventional approaches.}, number={24}, journal={ANALYTICAL CHEMISTRY}, author={Gach, Philip C. and Xu, Wei and King, Samantha J. and Sims, Christopher E. and Bear, James and Allbritton, Nancy L.}, year={2012}, month={Dec}, pages={10614–10620} }
 @article{detwiler_dobes_sims_kornegay_allbritton_2012, title={Polystyrene-coated micropallets for culture and separation of primary muscle cells}, volume={402}, ISSN={["1618-2642"]}, DOI={10.1007/s00216-011-5596-9}, abstractNote={Despite identification of a large number of adult stem cell types, current primary cell isolation and identification techniques yield heterogeneous samples, making detailed biological studies challenging. To identify subsets of isolated cells, technologies capable of simultaneous cell culture and cloning are necessary. Micropallet arrays, a new cloning platform for adherent cell types, hold great potential. However, the microstructures composing these arrays are fabricated from an epoxy photoresist 1002F, a growth surface unsuitable for many cell types. Optimization of the microstructures’ surface properties was conducted for the culture of satellite cells, primary muscle cells for which improved cell isolation techniques are desired. A variety of surface materials were screened for satellite cell adhesion and proliferation and compared to their optimal substrate, gelatin-coated Petri dishes. A 1-μm thick, polystyrene copolymer was applied to the microstructures by contact printing. A negatively charged copolymer of 5% acrylic acid in 95% styrene was found to be equivalent to the control Petri dishes for cell adhesion and proliferation. Cells cultured on control dishes and optimal copolymer-coated surfaces maintained an undifferentiated state and showed similar mRNA expression for two genes indicative of cell differentiation during a standard differentiation protocol. Experiments using additional contact-printed layers of extracellular matrix proteins collagen and gelatin showed no further improvements. This micropallet coating strategy is readily adaptable to optimize the array surface for other types of primary cells.}, number={3}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Detwiler, David A. and Dobes, Nicholas C. and Sims, Christopher E. and Kornegay, Joe N. and Allbritton, Nancy L.}, year={2012}, month={Jan}, pages={1083–1091} }
 @article{wang_jiang_sims_allbritton_2012, title={Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis}, volume={907}, ISSN={["1873-376X"]}, DOI={10.1016/j.jchromb.2012.09.003}, abstractNote={Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH2PO4 and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH2PO4 concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2 × 105 in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10−18–10−20 mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications.}, journal={JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES}, author={Wang, Kelong and Jiang, Dechen and Sims, Christopher E. and Allbritton, Nancy L.}, year={2012}, month={Oct}, pages={79–86} }
 @article{wang_shah_phillips_sims_allbritton_2012, title={Trapping cells on a stretchable microwell array for single-cell analysis}, volume={402}, ISSN={["1618-2650"]}, DOI={10.1007/s00216-011-5535-9}, abstractNote={There is a need for a technology that can be incorporated into routine laboratory procedures to obtain a continuous, quantitative, fluorescence-based measurement of the dynamic behaviors of numerous individual living cells in parallel, while allowing other manipulations, such as staining, rinsing, and even retrieval of targeted cells. Here, we report a simple, low-cost microarray platform that can trap cells for dynamic single-cell analysis of mammalian cells. The elasticity of polydimethylsiloxane (PDMS) was utilized to trap tens of thousands of cells on an array. The PDMS microwell array was stretched by a tube through which cells were loaded on the array. Cells were trapped on the array by removal of the tube and relaxation of the PDMS. Once that was accomplished, the cells remained trapped on the array without continuous application of an external force and permitted subsequent manipulations, such as staining, rinsing, imaging, and even isolation of targeted cells. We demonstrate the utility of this platform by multicolor analysis of trapped cells and monitoring in individual cells real-time calcium flux after exposure to the calcium ionophore ionomycin. Additionally, a proof of concept for target cell isolation was demonstrated by using a microneedle to locally deform the PDMS membrane in order to retrieve a particular cell from the array.}, number={3}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Wang, Yuli and Shah, Pavak and Phillips, Colleen and Sims, Christopher E. and Allbritton, Nancy L.}, year={2012}, month={Jan}, pages={1065–1072} }
 @article{wang_shah_phillips_sims_allbritton_2012, title={Trapping cells on a stretchable microwell array for single-cell analysis (vol 402, pg 1065, 2012)}, volume={404}, number={8}, journal={Analytical and Bioanalytical Chemistry}, author={Wang, Y. L. and Shah, P. and Phillips, C. and Sims, C. E. and Allbritton, N. L.}, year={2012}, pages={2519–2519} }
 @article{wang_balowski_phillips_phillips_sims_allbritton_2011, title={Benchtop micromolding of polystyrene by soft lithography}, volume={11}, number={18}, journal={Lab on a Chip}, author={Wang, Y. L. and Balowski, J. and Phillips, C. and Phillips, R. and Sims, C. E. and Allbritton, N. L.}, year={2011}, pages={3089–3097} }
 @article{phillips_lai_johnson_sims_allbritton_2011, title={Continuous analysis of dye-loaded, single cells on a microfluidic chip}, volume={11}, number={7}, journal={Lab on a Chip}, author={Phillips, K. S. and Lai, H. H. and Johnson, E. and Sims, C. E. and Allbritton, N. L.}, year={2011}, pages={1333–1341} }
 @article{grohman_kottegoda_gorelick_allbritton_weeks_2011, title={Femtomole SHAPE Reveals Regulatory Structures in the Authentic XMRV RNA Genome}, volume={133}, ISSN={["1520-5126"]}, DOI={10.1021/ja2070945}, abstractNote={Higher-order structure influences critical functions in nearly all noncoding and coding RNAs. Most single-nucleotide resolution RNA structure determination technologies cannot be used to analyze RNA from scarce biological samples, like viral genomes. To make quantitative RNA structure analysis applicable to a much wider array of RNA structure-function problems, we developed and applied high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to structural analysis of authentic genomic RNA of the xenotropic murine leukemia virus-related virus (XMRV). For analysis of fluorescently labeled cDNAs generated in high-sensitivity SHAPE experiments, we developed a two-color capillary electrophoresis approach with zeptomole molecular detection limits and subfemtomole sensitivity for complete SHAPE experiments involving hundreds of individual RNA structure measurements. High-sensitivity SHAPE data correlated closely (R = 0.89) with data obtained by conventional capillary electrophoresis. Using high-sensitivity SHAPE, we determined the dimeric structure of the XMRV packaging domain, examined dynamic interactions between the packaging domain RNA and viral nucleocapsid protein inside virion particles, and identified the packaging signal for this virus. Despite extensive sequence differences between XMRV and the intensively studied Moloney murine leukemia virus, architectures of the regulatory domains are similar and reveal common principles of gammaretrovirus RNA genome packaging.}, number={50}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Grohman, Jacob K. and Kottegoda, Sumith and Gorelick, Robert J. and Allbritton, Nancy L. and Weeks, Kevin M.}, year={2011}, month={Dec}, pages={20326–20334} }
 @article{gach_wang_phillips_sims_allbritton_2011, title={Isolation and manipulation of living adherent cells by micromolded magnetic rafts}, volume={5}, number={3}, journal={Biomicrofluidics}, author={Gach, P. C. and Wang, Y. L. and Phillips, C. and Sims, C. E. and Allbritton, N. L.}, year={2011} }
 @article{huang_jiang_wang_wang_sims_allbritton_zhang_2011, title={Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives}, volume={401}, ISSN={["1618-2650"]}, DOI={10.1007/s00216-011-5257-z}, abstractNote={Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP2 derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.}, number={6}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Huang, Weigang and Jiang, Dechen and Wang, Xiaoyang and Wang, Kelong and Sims, Christopher E. and Allbritton, Nancy L. and Zhang, Qisheng}, year={2011}, month={Oct}, pages={1881–1888} }
 @misc{kovarik_allbritton_2011, title={Measuring enzyme activity in single cells}, volume={29}, ISSN={["1879-3096"]}, DOI={10.1016/j.tibtech.2011.01.003}, abstractNote={Seemingly identical cells can differ in their biochemical state, function and fate, and this variability plays an increasingly recognized role in organism-level outcomes. Cellular heterogeneity arises in part from variation in enzyme activity, which results from interplay between biological noise and multiple cellular processes. As a result, single-cell assays of enzyme activity, particularly those that measure product formation directly, are crucial. Recent innovations have yielded a range of techniques to obtain these data, including image-, flow- and separation-based assays. Research to date has focused on easy-to-measure glycosylases and clinically-relevant kinases. Expansion of these techniques to a wider range and larger number of enzymes will answer contemporary questions in proteomics and glycomics, specifically with respect to biological noise and cellular heterogeneity.}, number={5}, journal={TRENDS IN BIOTECHNOLOGY}, author={Kovarik, Michelle L. and Allbritton, Nancy L.}, year={2011}, month={May}, pages={222–230} }
 @article{shadpour_zawistowski_herman_hahn_allbritton_2011, title={Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors}, volume={696}, ISSN={["1873-4324"]}, DOI={10.1016/j.aca.2011.04.012}, abstractNote={Pallet arrays enable cells to be separated while they remain adherent to a surface and provide a much greater range of cell selection criteria relative to that of current technologies. However there remains a need to further broaden cell selection criteria to include dynamic intracellular signaling events. To demonstrate the feasibility of measuring cellular protein behavior on the arrays using high resolution microscopy, the surfaces of individual pallets were modified to minimize the impact of scattered light at the pallet edges. The surfaces of the three-dimensional pallets on an array were patterned with a coating such as fibronectin using a customized stamping tool. Micropatterns of varying shape and size were printed in designated regions on the pallets in single or multiple steps to demonstrate the reliability and precision of patterning molecules on the pallet surface. Use of a fibronectin matrix stamped at the center of each pallet permitted the localization of H1299 and mouse embryonic fibroblast (MEF) cells to the pallet centers and away from the edges. Compared to pallet arrays with fibronectin coating the entire top surface, arrays with a central fibronectin pattern increased the percentage of cells localized to the pallet center by 3-4-fold. Localization of cells to the pallet center also enabled the physical separation of cells from optical artifacts created by the rough pallet side walls. To demonstrate the measurement of dynamic intracellular signaling on the arrays, fluorescence measurements of high spatial resolution were performed using a RhoA GTPase biosensor. This biosensor utilized fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure localized RhoA activity in cellular ruffles at the cell periphery. These results demonstrated the ability to perform spatially resolved measurements of fluorescence-based sensors on the pallet arrays. Thus, the patterned pallet arrays should enable novel cell separations in which cell selection is based on complex cellular signaling properties.}, number={1-2}, journal={ANALYTICA CHIMICA ACTA}, author={Shadpour, Hamed and Zawistowski, Jon S. and Herman, Annadele and Hahn, Klaus and Allbritton, Nancy L.}, year={2011}, month={Jun}, pages={101–107} }
 @article{kovarik_lai_xiong_allbritton_2011, title={Sample transport and electrokinetic injection in a microchip device for chemical cytometry}, volume={32}, ISSN={["0173-0835"]}, DOI={10.1002/elps.201100234}, abstractNote={Abstract}, number={22}, journal={ELECTROPHORESIS}, author={Kovarik, Michelle L. and Lai, Hsuan-Hong and Xiong, Jessie C. and Allbritton, Nancy L.}, year={2011}, month={Nov}, pages={3180–3187} }
 @article{xu_herman_phillips_pai_sims_allbritton_2011, title={Selection and Separation of Viable Cells Based on a Cell-Lethal Assay}, volume={83}, ISSN={["1520-6882"]}, DOI={10.1021/ac1023974}, abstractNote={A method to select and separate viable cells based on the results of a cell-lethal assay was developed. Cells were plated on an array of culture sites with each site composed of closely spaced, releasable micropallets. Clonal colonies spanning multiple micropallets on individual culture sites were established within 72 h of plating. Adjacent sites were widely spaced with 100% of the colonies remaining sequestered on a single culture site during expansion. A laser-based method mechanically released a micropallet underlying a colony to segment the colony into two genetically identical colonies. One portion of the segmented colony was collected with 90% efficiency while viability of both fractions was 100%. The segmented colonies released from the array were fixed and subjected to immunofluorescence staining of intracellular phospho-ERK kinase to identify colonies that were highly resistant or sensitive to phorbol ester-induced activation of ERK. These resistant and sensitive cells were then matched to the corresponding viable colonies on the array. Sensitive and resistant colonies on the array were released and cultured. When these cultured cells were reanalyzed for phorbol ester-induced ERK activity, the cells retained the sensitive or resistant phenotype of the originally screened subcolony. Thus, cells were separated and collected based using the result of a cell-lethal assay as selection criteria. These microarrays enabling clonal colony segmentation permitted sampling and manipulation of the colonies at very early times and at small cell numbers to reduce reagent, time, and manpower requirements.}, number={1}, journal={ANALYTICAL CHEMISTRY}, author={Xu, Wei and Herman, Annadele and Phillips, Colleen and Pai, Jeng-Hao and Sims, Christopher E. and Allbritton, Nancy L.}, year={2011}, month={Jan}, pages={278–283} }
 @article{jiang_sims_allbritton_2011, title={Single-cell analysis of phosphoinositide 3-kinase and phosphatase and tensin homolog activation}, volume={149}, journal={Faraday Discussions}, author={Jiang, D. C. and Sims, C. E. and Allbritton, N. L.}, year={2011}, pages={187–200} }
 @article{lai_xu_allbritton_2011, title={Use of a virtual wall valve in polydimethylsiloxane microfluidic devices for bioanalytical applications}, volume={5}, number={2}, journal={Biomicrofluidics}, author={Lai, H. H. and Xu, W. and Allbritton, N. L.}, year={2011} }
 @article{phillips_kang_licata_allbritton_2010, title={Air-stable supported membranes for single-cell cytometry on PDMS microchips}, volume={10}, number={7}, journal={Lab on a Chip}, author={Phillips, K. S. and Kang, K. M. and Licata, L. and Allbritton, N. L.}, year={2010}, pages={864–870} }
 @article{xu_luikart_sims_allbritton_2010, title={Contact printing of arrayed microstructures}, volume={397}, ISSN={["1618-2650"]}, DOI={10.1007/s00216-010-3728-2}, abstractNote={A novel contact printing method utilizing a sacrificial layer of polyacrylic acid (PAA) was developed to selectively modify the upper surfaces of arrayed microstructures. The method was characterized by printing polystyrene onto SU-8 microstructures to create an improved substrate for a cell-based microarray platform. Experiments measuring cell growth on SU-8 arrays modified with polystyrene and fibronectin demonstrated improved growth of NIH 3T3 (93% vs. 38%), HeLa (97% vs. 77%), and HT1080 (76% vs. 20%) cells relative to that for the previously used coating method. In addition, use of the PAA sacrificial layer permitted the printing of functionalized polystyrene, carboxylate polystyrene nanospheres, and silica nanospheres onto the arrays in a facile manner. Finally, a high concentration of extracellular matrix materials (ECM), such as collagen (5 mg/mL) and gelatin (0.1%), was contact-printed onto the array structures using as little as 5 μL of the ECM reagent and without the formation of a continuous film bridge across the microstructures. Murine embryonic stem cells cultured on arrays printed with this gelatin hydrogel remained in an undifferentiated state indicating an adequate surface gelatin layer to maintain these cells over time.}, number={8}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Xu, Wei and Luikart, Alicia M. and Sims, Christopher E. and Allbritton, Nancy L.}, year={2010}, month={Aug}, pages={3377–3385} }
 @article{shadpour_allbritton_2010, title={In situ Roughening of Polymeric Microstructures}, volume={2}, ISSN={["1944-8252"]}, DOI={10.1021/am900860s}, abstractNote={A method to perform in situ roughening of arrays of microstructures weakly adherent to an underlying substrate was presented. SU8, 1002F, and polydimethylsiloxane (PDMS) microstructures were roughened by polishing with a particle slurry. The roughness and the percentage of dislodged or damaged microstructures was evaluated as a function of the roughening time for both SU8 and 1002F structures. A maximal RMS roughness of 7-18 nm for the surfaces was obtained within 15-30 s of polishing with the slurry. This represented a 4-9 fold increase in surface roughness relative to that of the native surface. Less than 0.8% of the microstructures on the array were removed or damaged after 5 min of polishing. Native and roughened arrays were assessed for their ability to support fibronectin adhesion and cell attachment and growth. The quantity of adherent fibronectin was increased on roughened arrays by two-fold over that on native arrays. Cell adhesion to the roughened surfaces was also increased compared to native surfaces. Surface roughening with the particle slurry also improved the ability to stamp molecules onto the substrate during microcontact printing. Roughening both the PDMS stamp and substrate resulted in up to a 20-fold improvement in the transfer of BSA-Alexa Fluor 647 from the stamp to the substrate. Thus roughening of micrometer-scale surfaces with a particle slurry increased the adhesion of biomolecules as well as cells to microstructures with little to no damage to largescale arrays of the structures.}, number={4}, journal={ACS APPLIED MATERIALS & INTERFACES}, author={Shadpour, Hamed and Allbritton, Nancy L.}, year={2010}, month={Apr}, pages={1086–1093} }
 @article{xu_sims_allbritton_2010, title={Microcup Arrays for the Efficient Isolation and Cloning of Cells}, volume={82}, ISSN={["1520-6882"]}, DOI={10.1021/ac100434v}, abstractNote={Arrays of transparent, releasable micrometer-scale structures termed "microcups" were created for the purpose of patterning and isolating viable cells from small cell samples. Cells were captured by the microcups without the need for barriers or walls on the intervening substrate. Furthermore, in contrast to prior methods for creating cell arrays with releasable elements, no chemical modification of the substrate was required. Individual microcups were released from the array using a pulsed laser at very low energy. Improvements in microcup design enabled cells in suspension to be loaded into the microcups with greater than 90% efficiency. Cells cultured within the microcups displayed 100% viability and were cultured over 4 days yielding colonies that remained sequestered within the microcups to generate pure clonal populations. Standard microscopic imaging was used to identify cells or colonies of interest, and the microcups containing these cells were then released and collected. Individual target cells isolated in this manner remained viable as demonstrated by clonal expansion of 100% of collected cells. Direct comparisons with cell isolation by fluorescence-activated cell sorting and magnetic-bead-based isolation systems demonstrated that the microcup cell isolation procedure yielded higher purity, yield, and viability than these standard technologies when separating samples with small numbers of cells. The power of this technique was demonstrated by the isolation of hematopoietic stem cells from a human bone marrow aspirate possessing only 4000 total cells.}, number={8}, journal={ANALYTICAL CHEMISTRY}, author={Xu, Wei and Sims, Christopher E. and Allbritton, Nancy L.}, year={2010}, month={Apr}, pages={3161–3167} }
 @article{wang_dhopeshwarkar_najdi_waterman_sims_allbritton_2010, title={Microdevice to capture colon crypts for in vitro studies}, volume={10}, number={12}, journal={Lab on a Chip}, author={Wang, Y. L. and Dhopeshwarkar, R. and Najdi, R. and Waterman, M. L. and Sims, C. E. and Allbritton, N.}, year={2010}, pages={1596–1603} }
 @article{jiang_sims_allbritton_2010, title={Microelectrophoresis platform for fast serial analysis of single cells}, volume={31}, number={15}, journal={Electrophoresis}, author={Jiang, D. C. and Sims, C. E. and Allbritton, N. L.}, year={2010}, pages={2558–2565} }
 @article{wang_phillips_xu_pai_dhopeshwarkar_sims_allbritton_2010, title={Micromolded arrays for separation of adherent cells}, volume={10}, number={21}, journal={Lab on a Chip}, author={Wang, Y. L. and Phillips, C. and Xu, W. and Pai, J. H. and Dhopeshwarkar, R. and Sims, C. E. and Allbritton, N.}, year={2010}, pages={2917–2924} }
 @article{pai_xu_sims_allbritton_2010, title={Microtable arrays for culture and isolation of cell colonies}, volume={398}, ISSN={["1618-2650"]}, DOI={10.1007/s00216-010-3984-1}, abstractNote={Cell microarrays with culture sites composed of individually removable microstructures or micropallets have proven benefits for isolation of cells from a mixed population. The laser energy required to selectively remove these micropallets with attached cells from the array depends on the microstructure surface area in contact with the substrate. Laser energies sufficient to release micropallets greater than 100 μm resulted in loss of cell viability. A new three-dimensional culture site similar in appearance to a table was designed and fabricated using a simple process that relied on a differential sensitivity of two photoresists to UV-mediated photopolymerization. With this design, the larger culture area rests on four small supports to minimize the surface area in contact with the substrate. Microtables up to 250 × 250 μm were consistently released with single 10-μJ pulses to each of the four support structures. In contrast, microstructures with a 150 × 150-μm surface area in contact with the substrate could not be reliably released at pulse energies up to 212 μJ. Cassie–Baxter wetting is required to provide a barrier of air to localize and sequester cells to the culture sites. A second asset of the design was an increased retention of this air barrier under conditions of decreased surface tension and after prolonged culture of cells. The improved air retention was due to the hydrophobic cavity created beneath the table and above the substrate which entrapped air when an aqueous solution was added to the array. The microtables proved an efficient method for isolating colonies from the array with 100% of selected colonies competent to expand following release from the array.}, number={6}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Pai, Jeng-Hao and Xu, Wei and Sims, Christopher E. and Allbritton, Nancy L.}, year={2010}, month={Dec}, pages={2595–2604} }
 @article{gach_sims_allbritton_2010, title={Transparent magnetic photoresists for bioanalytical applications}, volume={31}, ISSN={["1878-5905"]}, DOI={10.1016/j.biomaterials.2010.07.087}, abstractNote={Microfabricated devices possessing magnetic properties are of great utility in bioanalytical microdevices due to their controlled manipulation with external magnets. Current methods for creating magnetic microdevices yield a low-transparency material preventing light microscopy-based inspection of biological specimens on the structures. Uniformly transparent magnetic photoresists were developed for microdevices that require high transparency as well as consistent magnetism across the structure. Colloidal formation of 10 nm maghemite particles was minimized during addition to the negative photoresists SU-8 and 1002F through organic capping of the nanoparticles and utilization of solvent-based dispersion techniques. Photoresists with maghemite concentrations of 0.01-1% had a high transparency due to the even dispersal of maghemite nanoparticles within the polymer as observed with transmission electron microscopy (TEM). These magnetic photoresists were used to fabricate microstructures with aspect ratios up to 4:1 and a resolution of 3 μm. Various cell lines showed excellent adhesion and viability on the magnetic photoresists. An inspection of cells cultured on the magnetic photoresists with TEM showed cellular uptake of magnetic nanoparticles leeched from the photoresists. Cellular contamination by magnetic nanoparticles was eliminated by capping the magnetic photoresist surface with native 1002F photoresist or by removing the top layer of the magnetic photoresist through surface roughening. The utility of these magnetic photoresists was demonstrated by sorting single cells (HeLa, RBL and 3T3 cells) cultured on arrays of releasable magnetic micropallets. 100% of magnetic micropallets with attached cells were collected following release from the array. 85-92% of the collected cells expanded into colonies. The polymeric magnetic materials should find wide use in the fabrication of microstructures for bioanalytical technologies.}, number={33}, journal={BIOMATERIALS}, author={Gach, Philip C. and Sims, Christopher E. and Allbritton, Nancy L.}, year={2010}, month={Nov}, pages={8810–8817} }