Pd 1 clone j43

In order to obtain tumor infiltrating lymphocytes, tumors from third passage B6CaP cells grown in parallel were mechanically disintegrated and digested for 1 h at 37°C in RPMI media with 10 mg/ml of Collagenase IV and 1 mg/ml of DNase. After digestion, white blood cells were isolated from tumors using anti CD45 antibodies conjugated with magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s protocol. Tumor infiltrating leukocytes were characterized with antibodies against CD3 (clone 145-2C11, eBioscience), CD19 (clone 1D3, BD Biosciences), CD49b (clone DX5, BD Biosciences), CD4 (clone H129.19, BD Biosciences), CD8 (clone 53-6.7, BD Biosciences), PD-1 (clone J43, eBioscience), Tim-3 (clone RMT3-23, eBioscience), Lag-3 (clone C9B7W, eBioscience) and CTLA4 (clone UC10-4F10-11, BD Biosciences). Flow through cells not captured by bead selection were stained with PDL-1 antibody (clone MIH5, eBioscience). For viability staining we used Live/Dead Aqua (Life Technologies). Gating controls were prepared with Fluorescence Minus One (FMO) stains of splenocytes or isotype control for tumor cells. Data were analyzed with FlowJo software (TreestarInc, Ashland, OR, USA).