Anti human il 17a pe

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.

The PBMCs and lymph node cells were incubated in 96-well bottom plates at 2 × 10⁶ cells per well in RP10 media (RPMI, 10% heat-inactivated FBS) alone or with phorbol 12-myristate 13-acetate (PMA) (20 ng/mL) plus ionomycin (1 μg/mL) for 4 to 6 h at 37 °C in the presence of BFA (10 μg/mL). The cells were harvested, washed with PBS, stained for the surface phenotypic markers, and fixed at RT with 2% PFA. The cells were then permeabilized (0.01% saponin), and the intracellular cytokines were stained using anti-human IFN-γ-V450 (BD, 560371, B27), anti-human IL-17A-PE (BD, 560486, N49-653), and anti-human TNF-α-APC (eBioscience, 17-7349-82, MAB11). All samples were analyzed using a Beckman Gallios instrument. The data were analyzed using the Kaluza software (Beckman Coulter Inc., Brea, CA, USA). PMA (cat. 16561-29-8), ionomycin (cat. 10634), brefeldin A (BFA) (cat. B7651), bovine serum albumin, and NaN₃ were all purchased from Sigma-Aldrich (St. Louis, MO, USA).

Purified PBMCs (100 μl) were collected into polystyrene fluorescence-activated cell sorting (FACS) tubes (BD Pharmingen™). To detect Tregs, 1 × 10⁶ PBMCs were surface stained with anti-human CD4-FITC (clone RPA-T4) and BV-605 mouse anti-human CD25 (clone 2A3) conjugated antibodies for 30 min at room temperature. The antibodies were washed twice, and the samples were treated with a fixation and permeabilization working solution. For intracellular staining, the samples were stained with anti-human Foxp3-PE (clone 259D/C7) or mouse IgG1 κ isotype (clone MOPC-21). To estimate Th17 cells, PBMCs were stimulated with a cell stimulation cocktail (eBioscience™, Cat# 00-4975-93) in complete culture medium (RPMI 1640 supplemented with 10% FBS) over 8 h at 37°C in 5% CO₂. The cells were collected and surface stained with anti-human CD4-FITC or mouse IgG1 κ isotype control-FITC. After permeabilization, intracellular staining was performed with anti-human IL-17A-PE (BD, Cat#560438) or mouse IgG1 κ isotype control-BV421 (BD, Cat#562438) according to the manufacturer’s instructions. The plots showing CD25 vs. Foxp3 expression were obtained after gating CD3⁺ CD4⁺ T cells. The same gating criteria were used for the plots of CD4 vs. IL-17. Cells were measured using flow cytometry (BD celesta, USA). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).