Antihuman cd3 clone sk7

Before antibody staining, cells were stained with Fixable Viability Dye eFluor™ 780 (eBioscience) on ice for 30 min to exclude dead cells, followed by treatment with a human Fc receptor-binding inhibitor (eBioscience) for 15 min at room temperature. Surface proteins were stained on ice for 30 min with the following fluorochrome-conjugated antibodies: antihuman CD3 (clone SK7, eBioscience), anti-human CD4 (clone RPA-T4, BioLegend), and antihuman CD8 (clone RPA-T8, eBioscience). For intracellular staining, cells were fixed and permeabilised with a FoxP3 staining buffer kit (Thermo Fisher Scientific) and stained with the following fluorochrome-conjugated antibodies: antihuman interferon-γ (IFN-γ) (clone B27, BD Biosciences), and antihuman tumour necrosis factor-α (TNF-α) (clone Mab11, BD Biosciences). Flow cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter), and the results were analysed using the FlowJo software (Tree Star Inc.).

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.).