Cd8 bv605 rpa t8

The HLA-null K562 human chronic myelogenous leukemia cell line was purchased from ATCC (CCL-234) and cultured in cRPMI. Media was added every two days to maintain cell concentrations at 0.25×10⁶ − 1.0×10⁶ cells/mL. Cryopreserved PBMCs were thawed and pre-treated with IL-12 (1 ng/mL) and/or IL-18 (10 ng/mL) for 24 hours. K562 cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE) according to the manufacturer’s instructions and then, co-cultured in a 96-well round bottom plate with 10:1, 5:1, and 2.5:1 PBMC effector to K562 target ratios. After 2.5 hours of incubation at 37°C and 5% CO₂, cells were stained with Viability Mastermix. For effector phenotyping, cells were stained for surface [CD8-BV605 (RPA-T8), NKG2D-BV711 (1D11), and PD-1-AF647 (EH12.2H7) (all Biolegend)] and intracellular markers [Perforin-Pacific Blue (dG9; Biolegend), IFN-γ-PE-Cy7 (B27; BD Biosciences), Granzyme B-PE (GB11; BD Biosciences)] as described in section 2.2. Samples were collected on a BD Accuri C6 cytometer and analyzed using FlowJo software. Percent specific lysis was calculated by subtracting background lysis (Annexin V⁺PI⁺) in wells containing 0:1 PBMC to K562 cells from lysis in wells containing effector cells.