Fix perm medium a and b

All antibodies listed were obtained from eBioscience (San Diego, CA, USA). A total of 5 μL labeled antibodies was added in each procedure. Because the PMA culture stimulates PBMC, it produces endocytosis of CD4 on the cell surface, which interferes with CD4 flow cytometric staining. As a result, CD3⁺CD8⁻ T cells were employed to counter-select CD4⁺ T cells in this investigation. Stimulated and cultured mononuclear cells were collected, pre-incubated for 15 min with unlabeled isotype control Abs (IgG1 or IgG2b), and then incubated with anti-CD3-FITC, anti-CD8-PE, or anti-CD4-FITC and anti-CD25-PE. A parallel control group was treated with their isotype controls. Then, mononuclear cells were treated with 100 μL FIX & PERM medium A and B (Invitrogen). Then, anti-IL-17-APC or anti-FoxP3-APC was added to PBMCs. A parallel control group was treated with isotype controls. Both suspensions were incubated at 4 °C away from light for one night. The markers for Treg cells and Th17 cells were CD4⁺CD25⁺FoxP3⁺, CD4⁺IL17⁺(unstimulated with PMA), or CD3⁺CD8⁻IL17⁺ (stimulated with PMA), respectively.