Aqua live dead fixable staining reagent

Mice were perfused using ice-cold PBS and brainstem with cerebellum and spinal cords were collected. Tissues were cut into small pieces using scissors, followed by 30 min of digestion with 0.8 mg/mL collagenase D (Roche) and 0.5 mg/mL DNAse (Sigma). Remaining pieces of tissue were homogenized and filtered through a 100 μm-cell strainer. After washing, the cell suspension was loaded onto a continuous 30% Percoll (GE) gradient and centrifuged for 30 min at 15 000 × g. The myelin layer was removed carefully, and the remaining cell suspension spun down. Flow cytometric analysis was performed following standard methods. We purchased 30-F11 (CD45) and M1/70 (CD11b) antibodies from BD Biosciences. In all stainings, dead cells were excluded using an Aqua Live/Dead fixable staining reagent (Invitrogen), and absolute cell numbers were determinded using AccuCheck Counting Beads (Life Technologies).

Cells were treated with anti-mouse CD16/CD32 (BioLegend) to block Fc receptors before staining. Staining was performed in PBS solution containing 5% FCS. For all experiments, dead cells were excluded using an Aqua Live/Dead fixable staining reagent (Invitrogen, Waltham, MA, USA). Monoclonal antibodies CD4 (GK1.5), CD5 (53-7.3), CD8 (53-6.7), CD11b (M1/70), CD11c (N418), CD19 (6D5), CD45 (30F11), CD107a (1D4B), Cx3cr1 (SA011F11), Vβ5.1 (MR9-4), Vβ8 (F23.1), Eomes (Dan11Mag), TCRβ (H57-597), and MHC II I-A/I-E (M5/114.15.2) were obtained either from BD, BioLegend, or eBioscience (San Diego, CA, USA). For surface staining, cells were incubated with monoclonal antibodies for 30 min on ice. In the cases of Eomes intracellular staining for CNS cells, fresh isolated cells were used without re-stimulation. Flow cytometric analysis was carried out on using a FACS Canto II (BD) with a FACS Diva software, and data were analyzed using a FlowJo (Tree Star, Waltham, MA, USA) software V10.7.