Anti hla abc clone w6 32

Expanded TILs and autologous tumour cells were co-cultured at an E:T ratio of 1:1 (1 × 10⁵ target cells). Simultaneously, TILs were cultured alone (negative control) and with 50 ng/ml PMA and 1 µg/ml Ionomycin (positive control). The cultures were incubated at 37 °C, 5% CO₂, 95% humidity for 24 h, before the supernatant was harvested. Supernatants were analysed for IFNγ production using the IFNγ ELISA kit (Diaclone, France) according to the manufacturer’s instructions. Where MHC class I and class II blocking experiments were performed, tumour cells were incubated with 50 µg/ml of anti-HLA ABC (clone W6/32, Biolegend, UK) and/or anti-HLA DR DP DQ (clone Tu39, BD Biosciences, UK) for 45 min at 4 °C prior to setting up the co-culture.

Cells were trypsinized, washed with ice-cold phosphate buffered saline (PBS), and pelleted by centrifugation. Cell pellets were then resuspended in anti-HLA-ABC (clone W6/32, #311403, BioLegend) conjugated to fluorescein isothiocyanate (FITC) (300:1 dilution), anti-β2-microglobulin (B2M) (clone 2M2, #316304, Biolegend) conjugated to FITC (300:1 dilution), or isotype control antibodies. Cells were incubated for 30 min at 4°C. After washing, cells were analyzed using a FACScalibur flow cytometer and CellQuestTM Pro version 6.0 software (both from Becton-Dickinson and Co.).