Ebioscience intracellular staining kit

For proliferation measurements, CFSE or CTe450 labeled cells were stained with Zombie aqua fixable viability dye (Biolegend) for 15 minutes, with surface antibodies for 20 min, then washed and fixed in 1% paraformaldehyde before flow cytometric analysis. For apoptosis assessment of PBMC and T cells, after surface staining, cells were stained with Annexin V (R&D Systems) per manufacturer’s instructions and were run immediately without fixation or were fixed in 1% PFA diluted in Annexin V binding buffer, containing CaCl₂ to preserve Annexin V binding. To measure intracellular cytokines, cells were treated with GolgiPlug^™ (BD BioScience) for the last 5 hours of culture, then stained with fixable viability dye and surface antibodies as above. Cells were then permeabilized using Invitrogen eBioscience^™ intracellular staining kit per manufacturer’s protocol and stained for IFNγ, TNFα and IL-2. Flow cytometry was performed on a FACSymphony or LSR II (BD Biosciences); data were analyzed with FlowJo software v.10.7.2 (BD Biosciences).