Facs lyse fix

The 3 μL of anti-human IL2RG (BD Biosciences) or matched isotype control (BD Biosciences) was added to 100 μL of blood, incubated for 10 minutes at room temperature. Red blood cells were lysed with FACs Lsye (BD Biosciences) for 10 minutes and then washed with Cell Wash (BD Biosciences) before being fixed (FACsFix, BD Bioscience). In total, 10,000 lymphocytes were acquired (FACs Calibur, BD Biosciences) and analyzed (CellQuest Pro, BD Biosciences) For STAT5 phosphorylation assay, cells were left unstimulated or 1×10 4 IU/mL IL-2 (Chiron) or 50 ng/mL IL-7 or IL-15 (R&D systems) were added to 100 μL of whole blood and placed at 37°C for 10 minutes. Two milliliters of prewarmed FACs Lyse/Fix (BD Biosciences) were then added to the blood, mixed, and placed at 37°C for a 10 minutes. The cells were pelletted and washed once with (Invitrogen) containing 1% fetal calf serum (STAT wash). The cells were resupended in cold Perm Buffer III (BD Biosciences) and placed at 4°C for 30 minutes. The cells were then washed once with STAT wash before 5 μL of antibodies (STAT5-ptyr and CD4 PerCP [BD Biosciences]) were added, and the cells were incubated at room temperature for 30 minutes in the dark, washed with STAT wash, and fixed (FACsFix, BD Biosciences). In total, 10,000 lymphocyte events were acquired (FACs Calibur, BD Biosciences) and analyzed using Cell-Quest Pro software (BD Biosciences).