title: A method for high-throughput functional imaging of single cells within heterogeneous cell preparations.
abstract: Functional characterization of individual cells within heterogeneous tissue preparations is challenging. Here, we report the development of a versatile imaging method that assesses single cell responses of various endpoints in real time, while identifying the individual cell types. Endpoints that can be measured include (but are not limited to) ionic flux (calcium, sodium, potassium and hydrogen), metabolic responsiveness (NAD(P)H, mitochondrial membrane potential), and signal transduction (H2O2and cAMP). Subsequent to fluorescent imaging, identification of cell types using immunohistochemistry allows for mapping of cell type to their respective functional real time responses. To validate the utility of this method, NAD(P)H responses to glucose of islet alpha versus beta cells generated from dispersed pancreatic islets, followed by the construction of frequency distributions characterizing the variability in the magnitude of each individual cell responses were compared. As expected, no overlap between the glucose response frequency distributions for beta cells versus alpha cells was observed, thereby establishing both the high degree of fidelity and low rate of both false-negatives and false-positives in this approach. This novel method has the ability not only to resolve single cell level functional differences between cell types, but also to characterize functional heterogeneity within a given cell type.
authors.affiliation: UW Diabetes Institute, Department of Medicine, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Adam S
authors.initials: AS
authors.lastname: Neal
authors.affiliation: UW Diabetes Institute, Department of Medicine, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Austin M
authors.initials: AM
authors.lastname: Rountree
authors.affiliation: UW Diabetes Institute, Department of Medicine, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Jared R
authors.initials: JR
authors.lastname: Radtke
authors.affiliation: Department of Mechanical Engineering, University of Washington, Seattle, WA 98195, USA.
authors.forename: Jianzhu
authors.initials: J
authors.lastname: Yin
authors.affiliation: UW Diabetes Institute, Department of Medicine, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Schwartz
authors.affiliation: UW Diabetes Institute, Department of Medicine, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Christiane S
authors.initials: CS
authors.lastname: Hampe
authors.affiliation: Department of Mechanical Engineering, University of Washington, Seattle, WA 98195, USA.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Posner
authors.affiliation: UW Diabetes Institute, Department of Medicine, University of Washington, Seattle, WA, 98195, USA.|Institute for Stem Cell and Regenerative Medicine, Department of Pharmacology, WA 98195, USA.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Cirulli
authors.affiliation: UW Diabetes Institute, Department of Medicine, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Sweet
mesh_terms: D000818:Animals; D050416:Glucagon-Secreting Cells / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D007150:Immunohistochemistry; D050417:Insulin-Secreting Cells / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D009249:NADP / Q000032:analysis; D061848:Optical Imaging / Q000379:methods*; D017207:Rats, Sprague-Dawley; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D009249:NADP; D005947:Glucose