STAT Protein Phosphorylation in CD4+ T Cells using the CellStream flow cytometer

Flow cytometry is a powerful method to study the orchestrated response of immune cell subsets to stimulation by combining immunophenotyping markers with fluorescent antibodies specific to phosphorylated signaling molecules. However, because many of the phosphorylation events occur on weakly expressed proteins and with low stoichiometry, flow cytometers must have high sensitivity to track important changes in signaling magnitude. Flow cytometers with sensitive detection, such as the Amnis systems, can be used to study phosphorylation events that are key to understanding the processes required for vaccine and immunotherapy development. In this application note, we used the Amnis CellStream Flow Cytometer to monitor the phosphorylation of signal transducer and activator of transcription (STAT) proteins—transcription factors that migrate to the nucleus upon phosphorylation and alter gene expression. STAT3, STAT5, and STAT6, which have been associated with prognosis in cancer and other malignancies, were simultaneously monitored in CD4+ T cells.

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