Anti human ifn γ fitc

AnnexinV-FITC/PI Kit (KeyGen Biotechnology, Nanjing, China) was used to evaluate the effect of FICZ and ITE on the apoptosis of PBMCs. For analysis of the frequency of Th1 and Th17, the cells were stimulated by adding PMA (50 ng/mL, Sigma-Aldrich, St. Louis, MO) and ionomycin (1 ug/mL, Sigma) for 1 h at 37°C. Then, brefeldin A (10 ug/mL, Sigma) was added for another 4 h; the cells were fixed and permeabilized using the eBioscience Cytofix/Cytoperm kit according to the manufacturer's instructions and then incubated with CD3-(PerCP)-Cy5.5, anti-human CD8-APC, anti-human IL-17A-PE, and anti-human IFN-γ-FITC (BD Biosciences). For phosphorylated STAT staining, stimulated CD4⁺T cells were fixed with Fix buffer (BD Biosciences) for 10 min at 37°C, permeabilized with Perm buffer (BD Biosciences) for 30 min on ice, and stained with anti-human pSTAT3-PE, anti-human pSTAT4-Per-cy5.5, anti-human pSTAT4-Per-cy5.5, anti-human pSTAT5-PE, or isotype control mAbs (BD Biosciences). Flow cytometric analysis was performed on a FACScan flow cytometer (BD Biosciences) to measure mean fluorescence intensity (MFI). Results were expressed as the percentage difference compared with isotypic control (IC) using the formula [mean fluorescence intensity (MFI) of sample − MFI of IC]/MFI of IC.

3 × 10⁶ PBMCs were incubated for 6 h with or without peptide, PMA/Iono or media. 5 μg/mL brefeldin A (BD Biosciences) was added after 1 h. Cells were then stained with Fixable Viability Stain 510 (BD Bioscience) and permeabilizated with the Cytofix/Cytoperm kit (BD Biosciences) before staining with anti-human IFN-γ-FITC (Clone 4S.B3, BD Biosciences) mAb. Data were acquired on a LSRFortessa X-20 driven by FACSDiva software (BD Biosciences) and analyzed using FlowJo software (version 10.4).