Flow activated cell sorter facs

First, brain cortex was isolated from experimental mice on the ice. Secondly, cell suspension was prepared via gridding brain tissue through 70 μm filters. Then sing cells were centrifuged at 3000 g for 10 minutes at 4°C. After centrifugation, we removed the supernatants and washed the cells with 100 μl PBS, followed by centrifugation for 10 minutes at 1000 g. Cells were suspended by 100 μl PBS again, and incubated with primary antibodies of CX3CR1-PE (1:200, Biolegend, San Diego, CA), CD16/32-FITC (1:100, Ebioscience, Franklin Lakes, NJ), and CD206-APC (1:50, Biolegend) at room temperature for 15 minutes. These cells were then washed with 3 ml PBS 0.1% sodium azide and 5% fetal bovine serum, followed by centrifugation for 5 minutes at 300 g suspend the cells in PBS and analyzed immediately using a Flow Activated Cell Sorter (FACS; BD Bioscience).

Firstly, splenocytes were isolated from adult C57BL/6 and GFP C57BL/6 mice by pressing spleens through 70 μm filters (BD Bioscience, San Jose, CA, USA). Secondly, CD4⁺ T cells were sorted using Mouse CD4⁺ T cell Pre-enriched Kit (Stemcell, Vancouver, BC, Canada) via magnetic activated cell sorting (MACS). CD4⁺/CD25⁺ Tregs were then enriched from CD4⁺ T cells by Flow Activated Cell Sorter (FACS; BD Bioscience) after being labeled with PE anti-mouse CD25 (Biolegend, San Diego, CA, USA). Enrichment of CD4⁺/CD25⁺ Tregs was further confirmed by Foxp3 staining (Biolegend). These cells were then cultured in the presence of anti-mouse CD28 (2 μg/ml, Ebioscience) and recombinant mouse IL-2 (400 U/ml, Life Technologies, Grand Island, NY, USA) in cell culture plates pre-coated with anti-mouse CD3e (10 μg/ml, Ebioscience, Franklin Lakes, NJ, USA). Three days later, CD44 and CD62L (Biolegend) staining were performed to confirm Treg activation. At the same time, mRNA level of IL-10, TGF-β and epstein-barr virus induced gene-3 (ebi3) were evaluated by qRT-PCR.