Fitc cd4 okt4

FoxP3 levels in CD3⁺CD4⁺ cells from cryopreserved PBMCs were determined by flow cytometry. A live/dead Fixable Dead Cell Stain (Invitrogen) was used to exclude dead cells from the analysis, followed by incubation for 30 min on ice with AF700-CD3 (clone OKT3) and FITC-CD4 (OKT4) (eBiosciences). Cells were then washed, fixed and permeabilized before adding PE-FOXP3 (clone 236A/E7) (eBiosciences) for 30 min. Appropriate isotype controls were included in each experiment. Flow cytometric analyses were performed on a Fortessa flow cytometer (BD Biosciences) with a total of 50,000 events recorded for each sample through a live CD3⁺CD4⁺ lymphocyte gate. Analysis of flow cytometry data was performed using FlowJo software. Tregs were defined as CD3⁺CD4⁺FoxP3⁺ cells.

For analysis of surface markers, the single‐cell suspensions of PBMCs or TILs were labelled with fluorochrome‐conjugated monoclonal antibodies specific to the relevant targets for 30 min. The primary antibodies include: anti‐human PE/cy7‐CD3 (OKT3, eBioscience), FITC‐CD4 (OKT4, eBioscience), Alexa Fluor 700‐CD8 (HIT8a, BD), Brilliant Violet 421‐TIGIT (MBSA43, eBioscience), APC‐CD96 (NK92.39, eBioscience), PE‐CD226 (c11A8, BioLegend) and FITC‐CD69 (FN50, BioLegend). For the detection of intracellular markers, cells were cultured with PMA/ionomycin/brefeldin A (eBioscience) for 4 h and then were permeabilised using a fixation/permeabilization kit (eBioscience). The primary antibodies include: anti‐human APC/CY7‐IFN‐γ (4S.B3, eBioscience), Alexa Fluor 647‐TNF‐α (MAB11, eBioscience), Pacific Blue‐granzyme B (QA16A02, BioLegend), and PE‐Ki‐67 (Ki‐67, BioLegend). For mouse cell staining, the primary antibodies include: anti‐mouse APC/cy7‐CD45 (30‐F11, BioLegend), FITC‐CD3 (17A2, BioLegend), PE‐CD4 (GK1.5, BioLegend), Alexa Fluor 700‐CD8 (Mp6‐XT22, BioLegend), PE/cy7‐CD69 (H1.2F3, eBioscience), PE‐IFN‐γ (XMG1.2, eBioscience), Brilliant Violet 421‐TNF‐α (Mp6‐XT22, BioLegend), APC‐IL‐2(JES6‐5H4, BioLegend), APC‐NK1.1 (S17016D, BioLegend) and BV421‐Foxp3 (MF‐14, eBioscience).