Mononuclear cells were isolated as described (Lutz et al., 2017 (link)). Spinal cords from WT and het-cKO female mice 21 days postimmunization were homogenized between frosted glass slides. Mononuclear cells were isolated at the interphase of a 30%–70% Percoll gradient (GE Healthcare). Spleens were mechanically homogenized, and red blood cells lysed. Single-cell suspensions were restimulated for 6 hr in vitro with phorbol 12-myristate 13-acetate, ionomycin, brefeldin, and monensin (eBioscience). After Fc receptor blockade, cells were stained with e780 viability dye (eBioscience) and CD45-BV421 (BD Pharmingen). Cells were fixed, permeabilized, and stained for IL-17α-PE (phycoerythrin) and IFN-γ-allophycocyanin (APC) (BD Pharmingen). Unstained cells were used for single-channel compensation, isotype controls, and fluorescence-minus-one controls. Compensation and analysis was done with Kaluza software (Beckman Coulter).
For MHC Class II, astrocytes were stimulated with IFNγ for indicated times and doses, then detached using Acutase (Fisher Scientific) for 3 min at 37°C. Cells were centrifuged, resuspended in fluorescence activated cell sorting (FACS) buffer (PBS, 2 mM EDTA and 2 mg/ml BSA), then stained with anti-MHCII-APC (Miltenyi Biotec 130–112-388) for 30 min on ice. Cells were washed twice with FACS buffer then analyzed on a LSR Fortessa (BD Bioscience).
For MHC Class II, astrocytes were stimulated with IFNγ for indicated times and doses, then detached using Acutase (Fisher Scientific) for 3 min at 37°C. Cells were centrifuged, resuspended in fluorescence activated cell sorting (FACS) buffer (PBS, 2 mM EDTA and 2 mg/ml BSA), then stained with anti-MHCII-APC (Miltenyi Biotec 130–112-388) for 30 min on ice. Cells were washed twice with FACS buffer then analyzed on a LSR Fortessa (BD Bioscience).