Cd95 apc clone dx2

Leukocytes were isolated by lysing red cells with Ammonium Chloride (NH₄Cl, 0.149 mol/L; Riedel-de Haën, Sigma-Aldrich, Germany), then washed in PBS and used for phenotypic staining. Leukocytes were washed twice with PBS and then stained with conjugate anti-human monoclonal antibodies (mAbs): CD3 PerCP-Cy5.5 (clone UCHT-1), Vδ2 FITC (clone B6), CD56 FITC (clone NCAM16.2), CCR7 PE-CY7 (clone 3DI2), CD95 APC (clone DX2), CD69 APC-Cy7 (clone FN50), CD158a PE (clone HP-3E4) (BD Biosciences), CD16 PeCy7 (clone 3G8) (Beckman Coulter), and NKp46 APC (clone 9E2) (Milteny Biotech). Leukocytes were incubated with the cocktail of antibodies for 20 min at room temperature, then washed once with PBS and fixed with 8% Paraformaldehyde (PFA) for 30 min at room temperature in the pass through chamber. After fixation, cells were handled out the pass through and washed once with PBS. After washing, samples were immediately acquired with the GUAVA flow cytometer (BD Biosciences) in a BSL2 laboratory. A total of 100,000–200,000 events was acquired for each sample and analyzed with FlowJo 10.0 software (BD Biosciences). The gating strategy is shown in S1 Fig.

Frozen PBMCs were thawed in RPMI medium supplemented with 10% FBS, penicillin, and streptomycin in the presence of DNAse (Millipore Sigma) then rested overnight in X‐Vivo medium (Lonza) supplemented with 5% human male AB serum. 1–3 × 10⁶ PBMC were stained with a saturating concentration of the following antibodies: CD3 AF700 (clone SP34‐2, BD Biosciences), CD4 eFluor450 (clone OKT4, eBioscience), CD8β ECD (2ST8.5H7, Beckman Coulter), CD95 APC (clone DX2, BD Biosciences), and CD28 PercpCy5.5 (clone CD28.2, BioLegend) in 1XPBS, 2% FBS and next incubated with LIVE/DEAD^® Fixable Dead Cell Stain (ThermoFisher Sci), both for 30 min at 4°C. Flow cytometry acquisition was performed on a BD Bioscience Fortessa and analyzed using FlowJo software (Tree Star).