Pe anti cd140a

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.

Cells were stained in the dark on ice for 15 minutes with flow cytometry antibodies. Cells were then washed once with 1X PBS and resuspended in 1X PBS for sorting as described previously (Mayo et al., 2014 ; Rothhammer et al., 2016 ). Antibodies used in this study were: PE anti-mouse CD45R/B220 (#553089, BD Biosciences, 1:100), PE anti-mouse TER-119 (#116207, Biolegend, 1:100), PE anti-O4 (#FAB1326P, R&D Systems, 1:100), PE anti-CD105 (#12–1051-82, eBioscience, 1:100), PE anti-CD140a (#12–1401-81, eBioscience, 1:100), PE anti-Ly-6G (#127608, Biolegend, 1:100), PerCP anti-Ly-6C (#128028, Biolegend, 1:100), APC anti-CD45 (#17–0451-83, eBioscience, 1:100), APC-Cy7 anti-CD11c (#561241, BD Biosciences, 1:100), and PE-Cy7 or FITC anti-CD11b (#25–0112-82, #11–0112-85, eBioscience, 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.