Tim 3 clone f38 2e2

For surface marker staining, 1 × 10⁵ T cells were seeded in 96-well plates with indicated target cells (unirradiated) at a 2:1 E:T ratio unless otherwise noted. Experiments with 2.5 × 10⁵ T cells were performed in 24-well plates. When indicated, γ-irradiated (100 Gy) K562 targets were used. When indicated, CD28 monoclonal antibody (clone CD28.2; eBiosciences) was applied at 10 μg/mL to provide CD28 costimulatory signal. Cell mixtures were incubated at 37 °C, and analyzed at the indicated time points with fluorescently labeled monoclonal antibodies binding CCR7 (clone REA108), CD19 (clone LT19), CD25 (clone BC96), CD27 (clone M-T271), CD45RA (clone T6D11), CD57 (clone TB03), PD-1 (PD1.3.1.3), PD-L1 (clone 29E.2A3), and Tim-3 (clone F38-2E2) (BioLegend and Miltenyi Biotec). V500-conjugated Annexin V (BD Biosciences) and Pacific Blue-conjugated Annexin V (BioLegend) were used to detect pre-apoptotic cells. CAR expression was probed with Protein L (Genscript) followed by PE-conjugated streptavidin (Jackson Immunoresearch) or with APC-conjugated polyclonal antibody binding human IgG Fcγ (Jackson Immunoresearch). EGFRt expression was probed with biotinylated Erbitux followed by PE-conjugated streptavidin. Analyses were performed on a MACSQuant VYB flow cytometer (Miltenyi Biotec) equipped with 405-, 488-, and 561-nm lasers. Data were processed using FlowJo software (TreeStar).