Analysis of Signaling Events by Dynamic Phosphoflow Cytometry

1Institut National de la Santé et de la Recherche Médicale, Unité 891, Centre de Recherche en Cancérologie de Marseille, F-13009 Marseille, France.
Science Signaling (Impact Factor: 6.28). 02/2009; 2(86):pl3. DOI: 10.1126/scisignal.286pl3
Source: PubMed


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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    • "). Intracellular staining for phosphoflow analysis was performed as previously described (Firaguay & Nunès, 2009). Cells were incubated 30 min at 4°C by adding: i) directly coupled antibodies: anti-phospho-Akt S473 Alexa Fluor 647 (Beckman Coulter, #A88915), anti-phospho-ERK-1/2 T202/Y204 Alexa Fluor 488 (Beckman Coulter, #A88928) and ii) Uncoupled antibodies: anti-phospho-STAT5 (Cell Signaling Technology, #9314). "
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