Anti mouse ifn γ percp cy5

The lymph node and spleen derived single cell suspensions were stimulated with 50 ng mL -1 PMA, 500 ng mL -1 ionomycin in the presence of 0.5 μL brefeldin A (to prevent the cells from secreting cytokines) (Biolegend, Switzerland) for 4 h at 37 °C, 5% CO 2 . The cell suspensions were washed with 0.5 mL of 1% fetal calf serum and centrifuged at 300 g for 5 min. Cells were stained according to manufacturer's instructions with antibodies for CD3 (PeCy7 anti-mouse CD3ε, Biolegend, CH) and CD4 (Pacific blue anti-mouse CD4, Biolegend, Switzerland). Following fixation with intracellular fixation buffer (Biolegend, CH) and permeabilization of the cells with Permeabilization buffer (Biolegend, CH), the lymph node cells were stained for IL-4 (anti-mouse IL-4 APC, eBiosciences, Switzerland), IL-10 (anti-mouse IL-10 PE, eBiosciences, CH), IL-17A (anti-mouse IL-17A Alexa Fluor 488, eBiosciences, Switzerland) and IFN-γ (anti-mouse IFN-γ PerCP-Cy5.5, eBiosciences, CH), according to manufacturer protocols. The cells were washed and re-suspended in MACS buffer (Mitenyi Coulter, Switzerland) and a Gallios flow cytometer (Beckman Coulter, Switzerland) was used to measure cellular fluorescence with data processing by Kaluza software (Beckman Coulter, Switzerland). The results were expressed as percentage of the total cell number.