Assessment of STAT5 phosphorylation by flow cytometry

IL2 responsive cells were starved by washing away IL2 used in culture and incubating at 37°C in complete media overnight. Cells were stimulated with increasing concentrations of IL2, ICp, IC35, or ICSK for 15 min at 37°C and then fixed with 1.5% paraformaldehyde (Polysciences, Inc., Cat.18814) for 10 min at room temperature. Cells were permeabilized with 100% ice-cold methanol (Fisher Scientific, Cat.A433P-4) for 30 min at 4°C and then washed and stained with FITC-labeled anti-STAT5-pY694 (Clone:SRBCZX, eBioscience, Cat.11-9010-42) for 30 min at room temperature. Intracellular staining was assessed by flow cytometry detection on the MACSQuant analyzer.