PT - JOURNAL ARTICLE AU - Firaguay, Guylène AU - Nunès, Jacques A. TI - Analysis of Signaling Events by Dynamic Phosphoflow Cytometry AID - 10.1126/scisignal.286pl3 DP - 2009 Sep 01 TA - Science Signaling PG - pl3--pl3 VI - 2 IP - 86 4099 - http://stke.sciencemag.org/content/2/86/pl3.short 4100 - http://stke.sciencemag.org/content/2/86/pl3.full SO - Sci. Signal.2009 Sep 01; 2 AB - 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.