Flow cytometry–based STAT phosphorylation assay

At the National Institutes of Health, PBMCs were thawed in XVIVO media (Lonza) with 10% human AB serum (Sigma-Aldrich), pelleted, washed with XVIVO, and resuspended in XVIVO media with 1% human AB serum at the concentration of 10⁶ cells/ml. Then the cells were stimulated with 1,000 U IL-2 (Peprotech) for 10 min at 37°C, fixed with BD Fix/Lyse buffer (BD Biosciences) for 10 min at 37°C, and then washed with cold PBS with 0.2% BSA. Next, the fixed cells were permeabilized with −20°C methanol for 20 min on ice, washed five times with cold PBS with 0.2% BSA, and then stained with surface and intracellular flow cytometry antibodies for 30 min at 4°C. The fixed, permeabilized, and stained cells were washed with PBS and resuspended in PBS with 0.2% BSA for flow cytometry analysis. In Newcastle, thawed PMBCs were rested for 4 h in serum-free RPMI media. After the addition of surface markers and a fixable viability dye, 2 × 10⁵ cells were stimulated for 10 min at 37°C with 100 ng/ml of IL-2, IL-7, or IL-15, or left unstimulated. The Transcription Factor Phospho Buffer set (BD Biosciences) was used to fix and permeabilize cells according to the manufacturer’s instructions. Cells were stained with the remaining surface as well as intracellular markers for 45 min at 4°C before cells were washed in TFP Perm/Wash buffer and finally resuspended in FACS buffer for acquisition.