Anti human tnf α fitc

NKG2A⁻ and NKG2A⁺ subpopulations were stimulated in U-bottomed 96-well plate (NUNC) with 400 ng/mL of plate-bound anti-CD3 mAb (produced by OKT3, ATCC CRL-8001, RRID:CVCL_2665) for 5 hours in the presence of 10 µg/mL of Brefeldin A (Sigma-Aldrich). After fixation with PBS 4% paraformaldehyde (VWR) for 10 minutes, cells were washed in PBS 0.1% BSA 0.1% Saponin (Sigma-Aldrich), centrifuged then stained with anti-human TNF-α-FITC (BioLegend, Mab11, RRID:AB_315258) and IFN-γ-APC (BioLegend, B27, RRID:AB_315443) or IL-2-APC (BioLegend, MQ1-17H12, RRID:AB_315098) mAbs for 30 minutes in PBS 0.1% BSA 0.1% Saponin. For the CD107a degranulation assay, cells were stimulated for 3 hours without Brefeldin A in the presence of anti-human CD107a-AF647 mAb (BioLegend, H4A3, RRID:AB_1227506) added at the beginning of stimulation. Stained cells were acquired in the singlet-cell gate on a BD FACSCanto II flow cytometer using the same software as above.