Pe anti mouse ter 119

Cells were stained in the dark on ice for 15 min with flow cytometry antibodies. Cells were then washed once with 1× PBS and resuspended in 1× PBS for sorting as described previously^{11 (link)–13 (link),73 (link)}. Antibodies used in this study were: PE anti-mouse CD45R/B220 (BD Biosciences, #553089, 1:100), PE anti-mouse TER-119 (Biolegend, #116207, 1:100), PE anti-O4 (R&D Systems, #FAB1326P, 1:100), PE anti-CD105 (eBioscience, #12–1051-82, 1:100), PE anti-CD140a (eBioscience, #12–1401-81, 1:100), PE anti-Ly-6G (Biolegend, #127608, 1:100), PerCP anti-Ly-6C (Biolegend, #128028, 1:100), APC anti-CD45 (eBioscience, #17–0451-83, 1:100), APC-Cy7 anti-CD11c (BD Biosciences, #561241, 1:100), and FITC anti-CD11b (eBioscience, #11–0112-85, 1:100). All cells were gated on the following parameters: CD105^negCD140a^negO4^negTer119^negLy-6G^negCD45R^neg. Astrocytes were subsequently gated on: CD11b^negCD45^negLy-6C^negCD11c^neg. Microglia were subsequently gated on: CD11b^highCD45^lowLy-6C^low. Pro-inflammatory monocytes were subsequently gated on: CD11b^highCD45^highLy-6C^high. Compensation was performed on single-stained samples of cells and an unstained control. Cells were sorted on a FACS Aria IIu (BD Biosciences). For sorting of TdTomato⁺ astrocytes, cells were sorted according to TdTomato fluorescence judged against a wild-type control animal using a yellow-green laser on a FACS Aria IIu.

Bone marrow cells obtained from tibias and femurs were isolated and stained using the following fluorescent conjugated antibodies: PE anti-mouse CD3, PE anti-mouse CD11b, PE anti-mouse CD45R/B220, PE anti-mouse CD45, PE anti-mouse Ter119 (mouse specific, no cross reactivity with human cells); appropriate isotype control antibodies were also used (all antibodies purchased from BioLegend). Cell fluorescence was measured immediately after staining (Becton Dickinson, FACS Calibur) and data were analyzed using the CellQuest software (Becton Dickinson). A maximum of 150,000 events were counted and final data were obtained within the lymphocyte gate.