Antifasl pe cy7 clone nok 1

Primary human CD3⁺ CD4⁺ CD27⁺ CD28⁺ T cells (10⁵) were activated with anti-CD3 (0.5 μg/mL) and recombinant human (rh) IL-2 (10ng/ml) for 10 days in the presence of 250-1,000 telomere vesicles (purified from either human or mouse CD3-depleted APC preparations as indicated), telomere depleted vesicles or left without any vesicle. Ten days later, the T cells were analysed on a Beckman Coulter flow cytometry with multiparametric settings. For the immunophenotype characterization, T cells were stained with anti-CD4 Alexa 700 (clone OKT4, Biolegend, Cat. 317626, 1:100), anti-CD27 PerCP5.5 (clone M-T271, Biolegend, Cat. 356408, 1:100), anti-CD28 APCVio770 (clone REA612, Miltenyi Biotec, Cat. 130-116-506, 1:100), anti-CD45RA BV421 (clone HI100, Biolegend, Cat. 3104130, 1:100), anti-CD45RO ECD (clone UCHL1, Beckman Coulter, Cat. M2712U, 1:100), anti-CD75 (Miltenyi Cat. 130113068), anti-PD1 APC (clone NAT105, Biolegend, Cat. 367406, 1:100), antiFasL PE-Cy7 (clone NOK-1, Biolegend, Cat. 306418, 1:100), anti-CTLA4 PE (clone L3D10, Biolegend, Cat. 349906, 1:100) and anti-TIM-3 VioBright FITC (clone REA602, Miltenyi Biotec, Cat. 130-109-711, 1:50). Cells were washed in PBS and analysed on a Cytoflex flow cytometer (Beckman Coulter).