Analysis of signaling events by dynamic phosphoflow cytometry.

Abstract : Many proteins involved in cell signaling are phosphorylated. To determine the phosphorylation status of these signaling molecules at the single-cell level, we present a protocol for using state-specific antibodies to detect target phosphoproteins with fluorescence measurements by flow cytometry. To improve the signal intensity, a sandwich-labeling method for the analysis of signaling proteins is performed. By comparing the phosphorylation state of proteins in the presence and absence of sodium pervanadate, a nonspecific tyrosine phosphatase inhibitor, we determined the relative amount of tyrosine-phosphorylated protein in the samples, which reflects the activity of the signaling pathway. This dynamic approach, in combination with the signal amplification through a sandwich-labeling method, produces accurate and reproducible measurement of the activity of signaling pathways.
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Science Signaling, American Association for the Advancement of Science, 2009, 2 (86), pl3. 〈10.1126/scisignal.286pl3〉
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Guylène Firaguay, Jacques Nunès. Analysis of signaling events by dynamic phosphoflow cytometry.. Science Signaling, American Association for the Advancement of Science, 2009, 2 (86), pl3. 〈10.1126/scisignal.286pl3〉. 〈inserm-00408711〉

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