Alexa fluor 647 anti mouse foxp3

Splenic CD4⁺ cells from approximately 28-day-old mice were obtained using the CD4⁺ T cell isolation kit (Miltenyi Biotec, 130-104-454) or by sorting with the BD FACSAria II cell sorter. CD4⁺ cells were processed using the Mouse Th1/Th2/Th17 (BD Biosciences, 560758) and Th17/Treg (BD Biosciences, 560767) phenotyping kits. Cells were cultured for 3–4 hours with or without 50 ng/mL PMA and 1 μg/mL ionomycin and then harvested for RNA-Seq or flow analysis. The following antibodies were used: PerCP-Cy5.5 anti–mouse CD4 (BD Biosciences, 550954), PE anti–mouse IL-17a (BD Biosciences, 559502), FITC anti–mouse IFN-γ (BD Biosciences, 554411), APC anti–mouse IL-4 (BD Biosciences, 554436), and Alexa Fluor 647 anti–mouse FOXP3 (BD Biosciences, 560402).

To determine the number of regulatory T cells in the splenocytes, flow cytometry was performed, as previously described [7 (link)]. The splenocytes were washed with PBS and stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (eBioscience, San Diego, CA, USA) and phycoerythrin (PE)-conjugated anti-mouse CD25 (eBioscience, San Diego, CA, USA). Next, the cells were fixed and stained with Alexa Fluor 647 anti-mouse Foxp3 (BD Biosciences, San Jose, CA, USA) overnight at 4 °C in the dark. After washing, the cells were analyzed. In order to measure Th1 and Th17 cells in the splenocytes, FITC-conjugated anti-mouse CD4 (eBioscience, San Diego, CA, USA), PE-conjugated anti-mouse IFN-γ (eBioscience, San Diego, CA, USA), and PerCP/Cyanine5.5-conjugated anti-mouse IL-17A (eBioscience, San Diego, CA, USA) were stained according to the supplier’s instructions.