Live dead fixable near ir dead cell marker

PBMCs from were thawed in R10 supplemented with DNaseI, washed twice by centrifugation (500 g) and rested for 2 hours at 37°C, 5% CO₂, followed by centrifugation and resuspension of the pellet in 50 μL antibody mixture for 20 minutes at room temperature. Cells were washed twice (PBS) and fixed with 2% PFA. Cells then acquired using an Aria Fusion Cytometer (BD) and analyzed with FlowJo Software, version 9.9.6 (Tree Star). The antibody cocktail consisted of LIVE/DEAD Fixable Near-IR Dead Cell Marker (Invitrogen, Thermo Fisher Scientific); anti–CD3 Brilliant Violet 785, clone OKT3; anti–γδ TCR PE, clone B1; anti–Vδ2 TCR PerCpCy5.5, clone B6; anti–Vγ9 TCR APC, clone B3; anti–Vα7.2 BV711, clone 3C10; anti–CD26 PE-Cy5 clone BA5b; anti–CD161 Brilliant Violet 605, clone HP3G10 (all from BioLegend); anti–CD45 V500, clone HI30 (BD); and anti–Vδ1 TCR FITC clone TS8.2 (Thermo Fisher Scientific).

PBMCs were thawed in RPMI media (RPMI 1640, 10% Heat inactivated FBS, 1% Penicillin/Glutamine, 1% L-glutamine) supplemented with DNase, washed twice by centrifugation (2,000 rpm for 5 min) and rested for 2 h at 37°C, 5% CO₂, followed by centrifugation and resuspension of the pellet in 50 μl antibody mixture for 20 min at room temperature. Cells were washed twice (PBS) and fixed with 2% PFA. Cells were then acquired using Aria Fusion III cytometer (BD) and analyzed with FlowJo software, version 10 (Tree Star). Cells collected from tissue were stained with same antibody mix consisting of: LIVE/DEAD Fixable Near-IR Dead Cell Marker (Invitrogen, Thermo Fisher Scientific); anti-CD45 V500, clone Hi30; anti-CD4 Brilliant Violet 496, clone SK3; anti-CD8 Brilliant Violet 395, clone RPA-T8 (All from BD); anti-CD3 Brilliant Violet 785, clone OKT3; anti- Vα7.2 Brilliant Violet 711, clone 3C10; anti-CD161 BV605, clone HP3G10; anti-CD26 PE-Cy5, clone BA5b; anti- γδ TCR PE, clone B1; anti-Vγ9 TCR APC, clone B3; anti-Vδ2 TCR PerCPCy5.5, clone B6 (All from BioLegend); anti-Vδ1 TCR FITC, clone TS8.2 (Thermo Fisher Scientific).