High-throughput signaling profiling in blood platelets by multiplexed phosphoflow cytometry

Multiplexed phosphoflow cytometry is a novel method that provides rapid and quantitative readouts on intracellular phosphoprotein signaling. In this approach, flow cytometry is combined with fluorescent cell barcoding (FCB) to facilitate high-throughput analyses of signaling events. After stimulation, fixed and permeabilized platelets are labeled with distinct dye intensities to create unique fluorescent signatures for individual samples. These uniquely labeled samples can be combined for simultaneous antibody staining and acquisition. During software analysis, multiplexed samples can be differentiated by their distinct fluorescence intensities and analyzed as if they had been acquired individually. Multiplexing eliminates intersample variation, increases statistical robustness, and allows 4–96 samples to be processed with no appreciable increase in antibody consumption or runtime. The method can be performed on washed platelets, platelet-rich plasma (PRP), and whole blood. Its inherent versatility can fulfil wide-ranging experimental requirements from simple dose titrations to complex pharmacologic screens.