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.

SUBMITTER: Ma H 

PROVIDER: S-EPMC3297669 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature