Fix and perm kit

T cells and Raji cells were plated at an E:T ratio of 1:1 (1 × 10⁵ effectors to 1 × 10⁵ targets) in a 96-well plate with U-shaped bottom. Cytokine exocytosis of T cells was blocked by supplementing protein transport inhibitor brefeldin-A (BFA, BD Biosciences, New York, USA) to the wells. The plate was incubated at 37˚C for 4 h. After that, cells were collected and then stained with surface markers (CD3, CD8 and CAR), followed by fixation and permeabilization utilizing a Fix and Perm kit (Biolegend). T cells were then stained with anti-IL-2, TNF-α, or/and IFN-γ flow antibodies at 4°C for 30 min in the dark. Stained cells were washed and resuspended in chilled PBS for flow cytometry.

The CD8-FITC, IFN-γ-APC-Cy7, CD4-FITC, CD25-APC, FoxP3-PE, lin1-FITC, CD11c-APC, HLA-DR-PE, PD-1-PE-cy7, and PD-L1-PE-cy7 mAbs were obtained from BioLegend (BioLegend, CA). Fix and Perm kit was obtained from BioLegend (BioLegend, CA). Intracellular cytokine production was detected by four-color flow cytometry [16 (link), 18 (link)], as described in previous papers. What need to be illustrated is that PMA-ionomycin stimulation causes the prominent alternation of cell membrane expression of CD4, so CD3⁺CD8⁻ cells are identified as CD4⁺ T cells [19 (link)]. After washing with PBS, the stained cells were analyzed by flow cytometry.