Fix perm solution

Cryopreserved PBMC were thawed in a 37°C water bath, immediately transferred to cell medium, washed, and resuspended in complete cell medium. Three million PBMC were stained in each test. Flow cytometry primary and secondary stainings were performed in flow buffer (PBS with 2 mM EDTA and 2% FBS) for 20 min in the dark at room temperature. After staining, cells were fixed for 15 min in the dark at room temperature using the Fix/Perm solution (eBioscience, USA). Finally, Fix/Perm solution was washed away, cells were resuspended in flow buffer, and kept at 4°C in the dark until they were acquired on the flow cytometer. For intracellular stainings, cells were permeabilized in Fix/Perm solution for 45 min and then stained for 30 min in permeabilization buffer (eBioscience, USA), diluted 1:10 with MQ water, before being washed and analyzed. All samples were run on an 18-color LSRFortessa (BD Biosciences, USA) equipped with 355, 405, 488, 561, and 639 nm lasers.

The peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density-gradient centrifugation to identify Th17 cells, defined as CD4 + T cells producing IL-17, and Treg cells, defined as CD4 + CD25 + FoxP3 + T cells. To avoid dead cells, surface and intracellular phenotyping of PBMCs was freshly performed with combinations of fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP) or allophycocyanin (APC)-labeled monoclonal antibodies (mAbs). For surface staining, after Fc-Receptor Blocking incubation, FITC conjugated mAbs against human CD4, and PE conjugated mAbs against human CD25 (all from BD Biosciences, San Jose, CA, United States) were used. The intracellular detection of IL-17/FoxP3 with PerCP-labeled anti-IL-17 (clone eBio64DEC17; eBioscience), APC-labeled anti-FoxP3 (clone 236A/E7; eBioscience) was obtained on cells fixed and permeabilized using Fix/Perm solution (eBioscience). For the detection of intracellular IL-17 production, PBMCs were stimulated for 4 h with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin in the presence of 10 μg/ml Brefeldin A (all from Sigma-Aldrich, St. Louis, MO, United States), and then stained with PerCP-labeled anti-IL-17 mAb. For gating strategy of the analysis of Th17 and Treg cells, see Figure 1.

Cells were stimulated with 50 ng/mL of phorbol myristate acetate (Sigma-Aldrich), 1 μg/mL of ionomycin (Sigma-Aldrich), and 10 μg/mL of GolgiStop (BD Biosciences) at 37°C under 5% (v/v) CO2 for 6 h, and then stained with fluorescein isothiocyanate-labeled anti-CD4 Abs (BD Biosciences). The cells were next fixed and permeabilized using a fix/perm solution (eBioscience) according to the manufacturer's instructions. The cells were next incubated with allophycocyanin (APC)-labeled IFN-γ Abs, APC-labeled IL-4 Abs and PE-labeled IL-17A Abs (BD Biosciences). Cells in the control group were stained with isotype control Abs. The samples were analyzed on a BD FACSCanto II flow cytometer (BD Biosciences); the data were evaluated using FlowJo software (ver. 7.6; TreeStar Inc., San Carlos, CA, USA).