Fixable live dead

Suspensions containing T cells were stained with fixable live/dead (Invitrogen) in PBS followed by surface antibody staining in FACS buffer (PBS with 0.5% BSA and sodium azide). For intracellular cytokine staining, cells were stained for intracellular molecules following fixation and permeabilization. For phosphostaining BD PhosFlow reagents were utilized and fixation/permeabilization protocols were carried according the to manufacturer’s protocol. After washing, cells were stained with antibody-fluorochrome conjugates for the indicated phosphorylated proteins (pZap70^Y319 (BD Biosciences), all other phospho-antibodies were purchased from Cell Signaling). Antibodies for surface staining and intracellular cytokine staining were purchased from BD Biosciences and eBiosciences. For determination of cytoplasmic membrane potential (V_m) cells were incubated in 2uM DiSBAC₄3 (Invitrogen) in conditions as indicated for 60 minutes prior to evaluation. For determination of [K⁺]i, cells were loaded with the potassium sensitive dye Asante Green-4 (TEFLabs) with PowerLoad (Invitrogen) per the manufacturer’s protocols. All experiments were conducted on a BD Fortessa flow cytometer (Becton Dickinson) and analyzed with FlowJo software.