Rat igg2b fitc isotype control

Splenic lymphocytes were adjusted to a concentration of 1 × 10⁶ cells per well and depleted of red blood cells via a 5-min incubation in NH₄Cl lysis buffer at 37 °C. Monoclonal antibodies coupled to fluorochromes specific for the following markers were used at a concentration of 1 μg/10⁶ cells: anti-mouse CD25-FITC, rat IgG1-PE isotype control, rat IgG2b-AF647 isotype control, and rat IgG2b-FITC isotype control (BD Pharmingen, San Diego, CA, USA). FoxP3, CD4, and IL-17 cells were stained using a commercial kit (BD Pharmingen, San Diego, CA, USA or eBiosciences, San Diego, CA, USA) according to manufacturer’s instructions. Appropriate positive, negative, and isotype controls were added to wells containing cells only. Treg subsets were quantified using a BD FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). 10,000 events were acquired for each sample. CD4+ lymphocytes in the lymphocyte fraction were gated, and the percentages of CD25 + foxP3+ cells, CD25 + foxP3- cells, IL-17⁺, and IL-17⁻ cells were calculated.

Splenic lymphocytes were adjusted to a concentration of 1 × 10⁶ cells per well and depleted of red blood cells via a 5-min incubation in NH₄Cl lysis buffer at 37 °C. Monoclonal antibodies coupled to fluorochromes specific for the following markers were used at a concentration of 1 μg/10⁶ cells: anti-mouse CD25-FITC, rat IgG1-PE isotype control, rat IgG2b-AF647 isotype control, and rat IgG2b-FITC isotype control (BD Pharmingen, San Diego, CA, USA). FoxP3, CD4, and IL-17 cells were stained using a commercial kit (BD Pharmingen, San Diego, CA, USA or eBiosciences, San Diego, CA, USA) according to manufacturer’s instructions. Appropriate positive, negative, and isotype controls were added to wells containing cells only. Treg subsets were quantified using a BD FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). 10,000 events were acquired for each sample. CD4+ lymphocytes in the lymphocyte fraction were gated, and the percentages of CD25 + foxP3+ cells, CD25 + foxP3- cells, IL-17⁺, and IL-17⁻ cells were calculated.

Splenic lymphocytes were adjusted to a concentration of 1 × 10⁶ cells per well and depleted of red blood cells via a 5-min incubation in NH₄Cl lysis buffer at 37 °C. Monoclonal antibodies coupled to fluorochromes specific for the following markers were used at a concentration of 1 μg/10⁶ cells: anti-mouse CD25-FITC, rat IgG1-PE isotype control, rat IgG2b-AF647 isotype control, and rat IgG2b-FITC isotype control (BD Pharmingen, San Diego, CA, USA). FoxP3, CD4, and IL-17 cells were stained using a commercial kit (BD Pharmingen, San Diego, CA, USA or eBiosciences, San Diego, CA, USA) according to manufacturer’s instructions. Appropriate positive, negative, and isotype controls were added to wells containing cells only. Treg subsets were quantified using a BD FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). 10,000 events were acquired for each sample. CD4+ lymphocytes in the lymphocyte fraction were gated, and the percentages of CD25 + foxP3+ cells, CD25 + foxP3- cells, IL-17⁺, and IL-17⁻ cells were calculated.