Anti ifnγ pe cy7 clone xmg1.2

Splenocytes were cultured in complete medium and stimulated with PMA (50 ng/ml), IO (500 ng/ml) and Brefeldin A (1 μg/ml) for 4–5 h at 37°C. Stimulated cells were washed, surface stained with anti-CD16/CD32, Aqua Live/dead fixable stain or Fixable Viability Dye eFluor 506, anti-CD3-v450 or anti-CD3 eFluor450, anti-CD4-FITC, APC-anti-CD44-APC eFluor780, for 20 min. Next, cells were fixed and permeabilized for 30 min. using the Foxp3 buffer set, followed by intracellular staining with anti-IL4-APC (clone 11B11; eBioscience) and anti-IFNγ-PE-Cy7 (clone XMG1.2; BD Pharmingen) for 30 min.

Cells from cLN were cultured in complete medium and stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and brefeldin A (1 μg/ml) for 4–5 h at 37°C. Stimulated cells were washed, surface stained with anti-CD16/CD32, Fixable Viability Dye eFluor 506, anti-CD3-eFluor450, anti-CD4-FITC, anti-CD44-APCeFluor780, for 20 min. Next, cells were fixed and permeabilized for 30 min using the Foxp3 buffer set, followed by intracellular staining with anti-IFNγ-PE-Cy7 (clone XMG1.2; BD Pharmingen) for 30 min.

Naïve synovial cells were used to analyze their Foxp3 expression profile, while the expression of IL-17A and IFNγ was assessed after in vitro stimulation of 8 × 10⁶ cells with 20 ng/ml PMA (Sigma-Aldrich) and 1 μg/ml Ionomycin (Sigma-Aldrich) in a 6-well flat-bottom plate (Falcon) for 6 h. To prevent cytokine GolgiPlug (BD) (1 μl/ml) and GolgiStop (0,6 μl/ml) (BD) were added after the first hour in culture. Afterwards cells were washed and stained for the expression of CD3 (anti-CD3-BV421, clone 17A2; BioLegend), CD4 (anti-CD4-APC-Fire, clone RM4-5; BioLegend), CD8 (anti-CD8-FITC, clone 53-6.7; BioLegend), CD19 (anti-CD19-PerCP Cy5.5, clone 6D5; BioLegend), CD25 (anti-CD25-PerCP Cy5.5, clone PC61; BioLegend), and living cells using L/D Aqua in BV510 (BioLegend) for 40 min at 4°C. Further, cells were prepared for intracellular staining using the Fixation/Permeabilization solution kit (Thermo Fisher Scientific). Antibodies for Foxp3 (anti-Foxp3 AF647, clone FJK-16s; eBioscience), IFNγ (anti-IFNγ-PE-Cy7, clone XMG1.2; BD), and IL-17A (anti-IL-17A-AF647, clone TC11-18H10.1; BioLegend) were diluted in permbuffer (Thermo Fisher Scientific) and applied to the cells for 30 min at 4°C. The flow cytometric analyses were performed using a cytofluorometer (FACS Canto II, BD) and data were evaluated using FCS-express 5 (De Novo Software).