Fitc conjugated anti il 17

Cell pellets were prepared from the spleens of spondyloarthritic SKG mice. To examine the population of T helper cells, the cells were stained with PerCP-conjugated anti-CD4 (catalog number 45-0042-82) and APC-conjugated anti-CD25 (catalog number 102012) antibodies (eBioscience; Biolegend, San Diego, CA, USA), and then permeabilized and fixed with CytoFix/CytoPerm (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. The cells were then further stained with PE-conjugated anti-FoxP3 (catalog number 12-5773-82), APC-conjugated anti-interferon (IFN) (catalog number 505810), and FITC-conjugated anti–IL-17 (catalog number 11-7177-81) (eBioscience; Biolegend, San diego, CA, USA).

To analyze intracellular cytokines, splenocytes were stained with PerCP-conjugated anti-CD4, APC-conjugated anti-CD25, FITC-conjugated anti-IL-17, and PE-conjugated anti-Foxp3 (eBiosciences), followed by fixation and permeabilization with a Foxp3 staining buffer kit (BD Bioscience) according to the manufacturer's instructions. Four hours before the staining, the cells were stimulated with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) (all from Sigma-Aldrich) and GolgiStop (BD Bioscience). All data was analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

To analyze intracellular cytokines, we stained splenocytes with PerCP-conjugated anti-CD4, APC-conjugated anti-CD25, FITC-conjugated anti-IL-17, and PE-conjugated anti-Foxp3 antibodies (eBiosciences), followed by fixation and permeabilization using a Foxp3 Staining Buffer Kit (BD Bioscience). Four hours before the staining, the cells were stimulated with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) (all from Sigma-Aldrich) and then treated with GolgiStop (BD Bioscience). All data were analyzed in the FlowJo software (Tree Star, Ashland, OR, USA).

For intracellular staining, cells were restimulated with 25 ng/mL phorbol 12-myristate 13-acetate and 250 ng/mL ionomycin (both from Sigma-Aldrich) for 4 h in the presence of GolgiStop (BD Biosciences, Sparks, MD, USA). Murine splenocytes were stained with surface PerCP-conjugated anti-CD4 (eBioscience) and APC-conjugated anti-CD25 (BioLegend, San Diego, CA, USA) antibodies. After fixation and permeabilization, cells were stained with FITC-conjugated anti-IL-17 or PE-conjugated anti-Foxp3 antibodies (eBioscience). Events were collected and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

For intracellular staining, cells were restimulated with 25 ng/mL phorbol 12-myristate 13-acetate and 250 ng/mL ionomycin (both from Sigma-Aldrich) for 4 h in the presence of GolgiStop (BD Biosciences, Sparks, MD, USA). Murine splenocytes were stained with surface PerCP-conjugated anti-CD4 (eBioscience) and APC-conjugated anti-CD25 (BioLegend, San Diego, CA, USA) antibodies. After fixation and permeabilization, cells were stained with FITC-conjugated anti-IL-17, APC-conjugated anti-IFN-γ, phycoerythrin (PE)-conjugated anti-IL-4, or PE-conjugated anti-Foxp3 antibodies (eBioscience). Human CD4⁺ T cells were stained with surface PE-Cy7-conjugated anti-CD4 and APC-conjugated anti-CD25 antibodies (BioLegend). After fixation and permeabilization, cells were stained with PE-conjugated anti-IL-17 or PE-conjugated anti-Foxp3 antibodies (eBioscience). Events were collected and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

Cells were stimulated with 25 ng ml⁻¹ PMA (Sigma-Aldrich, St Louis, MO), 250 ng ml⁻¹ ionomycin (Sigma-Aldrich) and Golgi Stop (BD Biosciences, San Diego, CA) in 5% CO₂ at 37 °C for 4 h. Cells were stained with Percp-conjugated anti-CD4 antibody and APC-conjugated anti-CD25 Ab (BD Pharmingen) and then stained with APC-conjugated anti-IFN-γ, PE-conjugated anti-IL-4, FITC-conjugated anti-IL-17 or PE-conjugated anti-Foxp3 (all from eBiosciences), followed by fixation and permeabilization using the Cytofix/Cytoperm Plus Kit (BD Biosciences) according to the manufacturer’s instructions. All samples were processed with FACSCalibur (BD Pharmingen), and the data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

We investigated changes in the population of Foxp3-positive Treg cells and Th17 cells after metformin treatment. To analyze intracellular cytokines, cells were stimulated with 25 ng/mL PMA and 250 ng/mL ionomycin (all from Sigma-Aldrich, St. Louis, MO, USA) and Golgi Stop (BD Biosciences, San Diego, CA, USA) in a 24-well plate and incubated for 4 h. Splenocytes were stained with Percp-conjugated anti-CD4 antibody, followed by fixation and permeabilization using the Cytofix/Cytoperm Plus Kit (BD Biosciences) according to the manufacturer's instructions and then stained with FITC-conjugated anti-IL-17 (all from eBiosciences). For analysis of Treg cells, splenocytes were surface labeled with CD4 and CD25, followed by fixation, permeabilization, and intracellular staining with Foxp3 as per the manufacturer's protocol. All samples were examined using a FACSCalibur (BD Pharmingen), and the data was analyzed using FlowJo software (Tree Star, Ashland, OR, USA).