2.4. Fluorescent activated cell sorting (FACS) analysis

Mice were terminally anesthetized with an overdose of sodium pentobarbital and transcardially perfused with heparinised PBS. Brain hemispheres were harvested in PBS 2%FCS 2 mM EDTA (FACS buffer), mechanically triturated and enzymatically dissociated using the Neural Tissue Dissociation Kit (P) (Miltenyi). Then, samples were passed through a cell strainer of 70 μm mesh (BD2 Falcon) with FACS buffer, and centrifuged twice at 500g for 10 min at 4 °C. After the second wash, cells were resuspended in 37% Percoll (GE Healthcare) and centrifuged at 500g for 30 min at 18 °C (Grabert et al., 2016, Pino and Cardona, 2011). The supernatant and myelin layers were discarded, and the cell pellet enriched with microglia/macrophages was resuspended in FACS buffer. Samples were split in several tubes and immunostained. Primary antibody labeling was performed for 1 h at 4 °C, using the following primary antibodies: rat-anti-mouse CD11b (BD Biosciences, clone M1/70), rat-anti-mouse CD45 (Biolegend, clone 30-F11), rat-anti-mouse CD3 (Biolegend, clone 17A2) and rat-anti-mouse Ly6C (BD Biosciences, clone AL-21), adding 7-Aminoactinomycin D (7-AAD, Biolegend) as a cell viability marker. Moreover, unstained cells and isotype-matched control samples were used to control for autofluorescence and/or non-specific binding of antibodies. In addition, blood samples were harvested by cardiac puncture and collected in EDTA tubes. Spleens were minced and passed through 70 um cell strainers. Leukocytes in blood and spleen were stained with rat-anti-mouse CD11b (BD Biosciences, clone M1/70), rat-anti-mouse CD45 (Biolegend, clone 30-F11), rat-anti-mouse CD3 (Biolegend, clone 17A2) and rat-anti-mouse Ly6C (BD Biosciences, clone AL-21) and Fixable Viability Dye eFluor™ 450 (eBioscience), using the same method described above. Erythrocytes were then lysed in RBC lysis buffer (eBioscience). Samples were run on a BD FACS Aria Flow cytometer. Data was analysed using FlowJo software.