PMA+Ionomycin (6 hours, in the presence of Monensin) stimulated human PBMCs were stained with CD4 Alexa Fluor® 647 and then treated Fixation Buffer (Cat 420801) followed by permeabilization with 1X Intracellular Staining Permeabilization Wash Buffer (Cat 421002) and stained with IFN-γ APC/Fire™ 750 for 30 min.

The vial of APC/Cy7 or APC/Fire™ 750 was stored in the dark at either 4°C or 37°C for 81 days. At certain intervals, an aliquot of reagent was used to stain Veri-Cells™, a lyophilized human PBMC product that effectively removes donor dependent variation in staining (cat. # 425002). Cells were stained for 15 min at room temp in cell staining buffer.

Human whole blood was stained for 20 min with CD3 (SK7) conjugates of APC/Fire™ 750, APC/Cy7, APC-H7, or APC-eFluor® 780 at each manufacturer's recommended optimal dilution, followed by RBC lysis and wash steps. Histograms were gated on lymphocyte populations based on forward and side scatter.

Human whole blood was stained for 20 min with CD3 (SK7) conjugates of APC/Fire™ 750, APC/Cy7, APC-H7, or APC-eFluor® 780 at each manufacturer's recommended optimal dilution, followed by RBC lysis and wash steps. Cells were then treated with either PBS control, 1% paraformaldehyde (PFA) in PBS, 4% PFA followed by 0.1% saponin, BioLegend's True-Nuclear™ Fix/Perm Buffer Set, BD's Transcription Factor Buffer Set or eBioscience's FoxP3/Transcription Factor Staining Buffer Set prior to analysis. Lymphocyte populations were gated on based on optimal forward and side scatter.

Human whole blood was stained for 20 min with CD3 (SK7) conjugates of APC/Fire™ 750, APC/Cy7, APC-H7, or APC-eFluor® 780 at each manufacturer's recommended optimal dilution, followed by RBC lysis and wash steps. Cells were then treated with either PBS control, 1% paraformaldehyde (PFA) in PBS, 4% PFA followed by 0.1% saponin, BioLegend's True-Nuclear™ Fix/Perm Buffer Set, BD's Transcription Factor Buffer Set or eBioscience's FoxP3/Transcription Factor Staining Buffer Set prior to analysis. Lymphocyte populations were gated on based on optimal forward and side scatter.