Anti cd57 apc

For flow cytometry analysis, cells were washed with PBS containing 10% FBS and incubated for 30 minutes at 4°C for labeling with anti-CD3-FITC (BD Biosciences, clone OKT3), anti-CD56-PE (Miltenyi Biotec, clone MEM-188), anti-CD57-APC (Biolegend, clone NK-1), anti-CD16-BV421 (BD Biosciences, clone 3G8), anti-NKG2C-BV510 (BD Biosciences, clone 134591) and 7AAD. For each sample, 100.000 events were recorded using a FACS Canto II cytometer (BD Bioscience). Cell populations were analyzed using FlowJo v.X.0.7 (TreeStar Inc.). Viability of thawed CB-NK cells was quantified using the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Invitrogen) following the manufacturer’s instructions.

The expression of immune exhaustion (PD-1) and senescence (CD57) markers was assessed as described previously (26 (link)). Briefly, after revival and resting overnight, PBMCs were resuspended in 1 ml PBS and then stained with violet amine reactive dye (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature for differentiation of dead and live cells. The samples having more than 90% viability were considered for further analysis. The cells were washed again and incubated with a cocktail of antibodies [anti-Vα24 PE, anti-Vβ11 FITC (Beckman Coulter, Marseilles, France), anti-CD57 APC (Biolegend, USA), anti-CD3 PETR (Invitrogen, Carlsbad, CA, USA), anti-CD3 APC, and anti-PD1 PerCpCy5.5 (BD Biosciences)] for 30 min at room temperature. After washing, the cells were fixed in 3% paraformaldehyde, acquired on FACSAria-I (BD Biosciences, USA) and analyzed using FACSAria-I (BD Biosciences, USA) and analyzed using FACSDiva software version 6.1.3 (BD Biosciences, USA).