title: High-throughput examination of fluorescence resonance energy transfer-detected metal-ion response in mammalian cells.
abstract: Fluorescence resonance energy transfer (FRET)-based genetically encoded metal-ion sensors are important tools for studying metal-ion dynamics in live cells. We present a time-resolved microfluidic flow cytometer capable of characterizing the FRET-based dynamic response of metal-ion sensors in mammalian cells at a throughput of 15 cells/s with a time window encompassing a few milliseconds to a few seconds after mixing of cells with exogenous ligands. We have used the instrument to examine the cellular heterogeneity of Zn(2+) and Ca(2+) sensor FRET response amplitudes and demonstrated that the cluster maps of the Zn(2+) sensor FRET changes resolve multiple subpopulations. We have also measured the in vivo sensor response kinetics induced by changes in Zn(2+) and Ca(2+) concentrations. We observed an ∼30 fold difference between the extracellular and intracellular sensors.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
authors.forename: Hairong
authors.initials: H
authors.lastname: Ma
authors.forename: Emily A
authors.initials: EA
authors.lastname: Gibson
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Dittmer
authors.forename: Ralph
authors.initials: R
authors.lastname: Jimenez
authors.forename: Amy E
authors.initials: AE
authors.lastname: Palmer
mesh_terms: D000465:Algorithms; D002118:Calcium / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007477:Ions / Q000737:chemistry; D015032:Zinc / Q000737:chemistry*
chemical_list: D007477:Ions; D015032:Zinc; D002118:Calcium