Anti human cd8 apc cy7 clone sk1

Cell surface expression of lymphocyte antigens was performed by monoclonal antibody staining of freshly isolated MMCs and PBMCs, followed by flow cytometry using a BDLSRII instrument (Becton-Dickinson, Palo Alto, California, United States) with analysis using FlowJo software (TreeStar). Monoclonal antibodies used in this study included: anti-human CD3-Pacific Blue (PB) (clone UCHT1) (BD Pharmingen), anti-human CD4-Alexa700 (clone RPA T4) ((BD Pharmingen), anti-human CD8 APC-Cy7 (clone SK1) (BD Pharmingen), anti-human CD38-PE (clone HIT2) (BD Pharmingen), and anti-human Ki67-PE (clone B56) (BD Pharmingen), and the appropriate isotype controls. Flow cytometric analyses were performed as previously described [3 (link)].

The CD4⁺ TIL clone was generated by limiting dilution and confirmed using the IO Test^® Beta Mark TCR V beta Repertoire Kit (Beckman Coulter, Brea, CA) by flow cytometry. A total of 2 × 10⁵ T cells were co-cultured with 4 × 10⁴ autologous tumour cells for 5 h at 37 °C (and 5% CO₂) in a 96-well tissue culture plate containing 200 μl assay medium/well (RPMI 1640 with 10% FBS and penicillin/streptomycin; both from Thermo Fisher Scientific, Waltham, MA). During the incubation period, 1.3 μg/ml of monensin (Merck KGaA, Darmstadt, Germany), and 4 μl of the anti-human CD107a-Alexa Fluor 700 antibody (Clone H4A3; BD Biosciences, Franklin Lakes, NJ) were added. PMA = phorbol 12-myristate 13-acetate (PMA) was used as the positive control and assay medium alone without tumour cells was used as negative control. After 5 h of incubation, the cells were stained with anti-human CD3-PE/Cy7 (Clone HIT3A; BioLegend, San Diego, CA), anti-human CD4-V450 (Clone RPA-T4) and anti-human CD8-APC/Cy7 (Clone SK1) (both from BD Biosciences, Franklin Lakes, NJ), and analysed by flow cytometry.