Percp anti mouse cd4 antibody

The spleen lymphocytes of EAE mice (on the 20th day after immunization) were separated with Ficoll density gradient centrifugation. Peritoneal macrophages were isolated as previously described (7 (link)), washed twice with FACS (containing 2% fetal bovine serum), then stained with APC anti-mouse F4/80 antibody (BioLegend, 123116) and PE anti-mouse MHC-II antibody (BioLegend, B117132), incubated at 37°C for 30 min, and then washed once with FASC and resuspended in 1% paraformaldehyde. For staining of spleen cells, firstly, the cells were incubated with cell stimulation cocktail plus protein transport inhibitors (Invitrogen, 2260623) for 4–6 h, washed with FACS once for staining with PerCP anti-mouse CD4 antibody (BioLegend, 100538), and then washed with FACS; 200 μl fixation/permer buffer (Invitrogen, 2220750) was added at 4°C overnight, and then permer buffer dilution washing and staining were done on the APC anti-mouse IFN-γ antibody (BioLegend, 505810), PE anti-mouse IL-17 antibody (BioLegend, 506904), and APC anti-mouse Foxp3 antibody (eBioscience, E07303-1635), and finally washing with permer buffer and resuspension were performed.