Antihuman tim3 clone f38 2e2

All samples were analyzed using a NovoCyteTM (ACEA Biosciences), an LSR Fortessa, or a C6 flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA). The antibodies used included antihuman TIM3 (clone F38-2E2); antihuman LAG3 (clone 11C3C65); antihuman PD-1 (clone NAT105); antihuman CD3 (clone UCHT1); antihuman CD4 (clone OKT4); antihuman CD8 (clone OKT8); antimouse immunoglobulin G2a (IgG2a) isotype control-APC (clone RMG2a-62); antimouse IgG1κ isotype control-PE; antimouse IgG1κ isotype control-PerCP/Cy5.5; and antimouse IgG1κ isotype control-APC (clone MOPC-21) (BioLegend, San Diego, CA, USA). All flow-cytometry-related staining procedures were performed on ice for 30 min, and the cells were washed with PBS containing 1% FBS before cytometry analysis. PB and tumor samples from mouse xenografts were treated with red blood cell lysis buffer (BioLegend), and the cells were stained with the corresponding antibodies.

Flow cytometry analysis was performed as previously described [16 (link), 17 (link)]. Briefly, single-cell suspensions were stained with the following fluorochrome-conjugated antibodies: anti-human CD3 (clone SK7, eBioscience), anti-human CD8 (clone RPA-T8, eBioscience), anti-human PD-1 (clone EH12.2H7, BioLegend), anti-human TIM-3 (clone F38-2E2, BioLegend), and anti-human TIGIT (clone A15153G,. BioLegend). For intracellular staining, cells were fixed and permeabilized using a FoxP3 staining buffer kit (Thermo Fisher Scientific) and stained with the following antibodies: anti-human Ki67 (clone Ki67, BioLegend), anti-human interferon-γ antibody (clone B27, BD Bioscience), and anti-human tumor necrosis factor (TNF)-α antibody (clone Mab11, BD Bioscience). All flow data were acquired using a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJo software (Tree Star Inc.).