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.