Il 17a tc11 18h10

Single-cell suspensions were prepared as described above. Cells were stimulated for 4 h with 50 ng/mL of PMA (Sigma-Aldrich, St. Louis, MO, USA) and 500 mg/mL of ionomycin (Sigma-Aldrich, St. Louis, MO, USA) in the presence of GolgiStop (BD Biosciences, San Diego, CA, USA). After stimulation, the cells were washed and resuspended in a staining buffer, where they were stained with anti-mouse CD3 (145-2C11, BD Bioscience, San Jose, CA, USA), CD4 (GK1.5, Biolegend, San Diego, CA, USA), and CD8 (53-6.7, BD Bioscience, San Jose, CA, USA) antibodies and incubated for 30 min at 4 °C in PBS with 2% FBS. Cells were then fixed and permeabilized with the Cytofix/Cytoperm Solution Kit (BD Biosciences, San Jose, CA, USA) and stained with anti-mouse IFN-γ (XMG1.2, Biolegend, San Diego, CA, USA), IL-17A (TC11-18H10, Biolegend, San Diego, CA, USA), IL-17F (O79-289, Biolegend, San Diego, CA, USA), and GM-CSF (MP1-22E9, Biolegend, San Diego, CA, USA) antibodies for 1 h at room temperature. The results were analyzed using a BD LSRFortessa Cell Analyzer and BD FlowJo (Tree Star Inc., Oakland, CA, USA).