Anti human cd3 brilliant violet 421

PBMCs (3 × 10⁵) were pretreated with TOFA (0.08–2 μM) and cultured in the presence of anti-CD3/CD28 in a 1:1 ratio for 2 days. Then the cells were stimulated using IL-2 (200 U/mL) for 30 minutes. The cells were fixed for 20 minutes at RT with 4% paraformaldehyde. Washed cells were permeabilized in ice-cold 75% methanol for 30 minutes and simultaneously incubated with the following antibodies: anti-human CD3 Brilliant Violet 421, CD8 PE-Cy7, p-STAT1 (Tyr701) PE, and p-STAT3 (Tyr705) Alexa Fluor 488 (BioLegend). Flow cytometry analysis was performed using a BD Biosciences LSR Fortessa flow cytometer. Ultimately, the harvested data were analyzed with FlowJo software (version 10.0 TreeStar). STAT phosphorylation was appraised in CD3⁺ and CD8⁺ TCs.

After isolation, PBMCs (3 × 10⁵) were cultured in the presence of anti-CD3/CD28 in a 1:1 ratio and treated with TOFA (0.08–2 μM) for 2 days. To investigate the intercellular cytokines in TCs, hemocytes were stimulated by a leukocyte activator, incorporating ionomycin (500 ng/mL; MilliporeSigma) and PMA (50 ng/mL; MilliporeSigma), then inhibited by brefeldin A (1 μg/mL; MilliporeSigma) in a 5% CO₂ environment at 37°C for 4 hours. Thereafter, processed cells were stained with anti-human CD3 Brilliant Violet 421 (BioLegend), anti-human CD8 PE-Cy7 (BioLegend), and anti-human IFN-γ APC (BD Biosciences) for Th1 cell analysis; anti-human IL-17A PE (BioLegend) for Th17 cell analysis; and anti-human FOXP3 PE (BioLegend) for Treg analysis. The labeled cells were measured using a BD Biosciences LSR Fortessa flow cytometer. Ultimately, the harvested data were analyzed with FlowJo software (version 10.0; TreeStar).