Cd69 fitc

The cell surface markers and experimental methods for mouse brain microglia were assessed as described previously (Frank et ). Briefly, when the mice were sacrificed, one side of the hippocampus was quickly stripped on ice and placed in a MACS tube to prepare a single-cell suspension. The supernatant was collected and mixed with myelin removal beads to enrich the microglia. The cells were stained with CD11b-FITC (cat.no.553310, BD Pharmingen, San Diego, CA, USA) CD86-PE (cat.no.4325501, Invitrogen, Carlsbad, CA, USA), and CD45-APC antibodies (cat.no.103112, BioLegend, San Diego, CA, USA). Peripheral blood, spleen, and lymphoid single-cell suspensions were incubated with erythrocyte lysate. Then, T cells were evaluated by staining with CD69-FITC (cat.no.104506, BioLegend), CD3-PE (cat.no.12-0031-82, eBioscience, San Diego, CA, USA), and CD4-PE-Cy5 (cat.no.100410, Biolegend) . B cell population was estimated by staining with CD69-FITC (cat.no.104506, BioLegend) and B220-PE (cat.no.12-0452-85, eBioscience). T helper 17 (Th17) were incubated with 5 ng/ml PMA and 1 ng/ml ionomycin for 5 h. After 30 min, brefeldin A was added at a final concentration of 10 ng/ml. After washing, cells were stained with CD4-FITC (cat.no.561835, BD Biosciences, San Jose, CA, USA) and IL-17A-PE (cat.no.506903, Biolegend).