Fluorochrome conjugated antibodies against cd3

Tumor cells expressing PD-L1 were stained with fluorochrome-conjugated antibodies against PD-L1 (BD Biosciences, USA) markers. CAR T cell lines were stained with fluorochrome-conjugated antibodies against the following markers: CD3, CD8, PD-1, TIM-3, LAG-3, CD25, CD69, CD45RO, CD62L, and CCR7 (BD Biosciences, USA). For assessment of the active promoter, CAR T cells transfected with the EGFP receptor gene were directly measured using flow cytometry. CAR expression was detected using an indirect method with biotinylated MSLN protein and streptavidin-coupled phycoerythrin (PE) antibody (BD Biosciences, USA). For the PD-1-blocking assay, CAR T cells were respectively stained with fluorochrome-conjugated antibodies against CD3, CD8, PD-1, TIM-3, LAG-3, CD25, CD69, CD45RO, CD62L, and CCR7 (BioLegend, USA). For assessment of CAR T cell cytotoxic activity, 2 × 10⁵ CAR T cells (CD19 CAR T, pS338B-αPD-1/CAR T, or pS-CIFT-αPD-1/CAR T) and 2 × 10⁵ tumor cells (SKOV3-PD-L1-EGFP or SKOV3-EGFP) were incubated for 24 h. The cell mixture was stained with anti-CD3 and anti-PD-L1 and was measured using flow cytometry. All samples were acquired on the Navios flow cytometer (Beckman Coulter, USA) and analyzed with FlowJo software vX.0.7, Kaluza v2.0 software, and GraphPad Prism 8 (GraphPad, USA).