Cd3 percp cd4 fitc cd8 pe trucount kit

After centrifugation, cells were suspended and washed three times with FACS wash buffer (1 x PBS containing 0.5% BSA and 0.03% sodium azide). Then, cells were stained with an antibody directed against MUC1 (BD, San Jose, CA, USA). To detect phosphorylated STAT3 and STAT5 in T cells, anti-human pSTAT3 (eBioscience, Santiago, CA, USA) and anti-human pSTAT5 (eBioscience, Santiago, CA, USA) monoclonal antibodies were used for staining. After 5 weeks of co-incubation with target cells, the ligands of PD-1, TIM-3 and LAG-3 on CAR-T cells were detected by anti-human CD279 (BD, CA, USA), anti-human CD366 (eBioscience, Santiago, CA, USA) and anti-human CD223 (eBioscience, CA, USA) antibodies, respectively. Moreover, anti-human TNF-α (BD, CA, USA) and anti-human IFN-γ (BD Bioscience, Franklin, NJ, USA) antibodies were used to investigate the level of intracellular TNF-α and IFN-γ in CAR-T cells. In addition, the CD3-PerCP/CD4-FITC/CD8-PE TruCOUNT kit (BD Bioscience, Franklin, NJ, USA) was used to determine the number of human CAR-T cells in peripheral mouse blood. CD4 + and CD8 + T cells were quantified according to the manufacturer’s instructions.

The harvested cells were centrifuged, washed thrice with FACS wash buffer (1×PBS containing 0.5% BSA and 0.03% sodium azide), and stained with anti-EGFR-PE (BD, San Jose, CA, USA), anti-Fab-FITC (eBioscience, CA, USA), anti-CD3-FITC (eBioscience, CA, USA), anti-CD4-PE (BD, CA, USA), anti-CD8-APC (eBioscience, CA, USA) and anti-PD-1-PB450 (eBioscience, CA, USA) antibodies as appropriate. The intracellular IFN-γ in T cells was also stained using anti-IFN-γ-FITC antibody (BD, CA, USA). In addition, the peripheral blood of the tumor-bearing mice was stained with the CD3-PerCP/CD4-FITC/CD8-PE TruCOUNT kit (BD Bioscience), and the CD4+ and CD8+ T cells were quantified according to the manufacturer’s instructions.