Anti ly6g pe

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.

Mice were anesthetized with tribromoethanol, and perfused via the left
ventricle with cold PBS. Brains were then harvested and cells were collected
using the Miltenyi Neural dissociation kit (San Diego, CA). Cells were then
blocked with Anti CD16/CD32 (15 min, 4 °C). Cells were then stained with
the following monoclonal antibodies at 4 °C for 45 min: Anti-CD11b PE,
anti-CD8 Pac Blue, anti-CD19 Percp-cy5.5, anti-CD4 Pe-cy7, anti CD45
PE-Dazzle594, anti-CD86 FITC, anti-CD45 Alexa Flour 700, anti-Ly6G PE,
anti-CD11c Pacific Blue, anti-MHC II Percpcy5.5, anti-CD11b Bv650 all purchased
from Biolegend (San Diego, CA), and anti-Ly6C PEtxRed (BD Biosciences, San Jose,
CA), anti-F4/80 APC-cy7, anti-O4 Alexa Flour 488 (R & D Systems),
anti-ASCA-2 APC (Miltenyi), anti-CD3 V500 (BD Biosciences), and anti-CD192 APC
(R & D Systems, Minneapolis, MN). Cells were then fixed, permeabilized,
(Fixation Buffer and Permeabilization wash buffer; Biolegend) and stained with
intracellular stains anti-CD68 PEcy7 (Biolegend), anti-NeuN Alexa Flour 700
(Novus Biologicals, Littleton, CO) (4 °C 45 min). Cells were then washed
twice and resuspended. Samples were run on an LSRFORTESSA flow cytometer (BD
Biosciences). Data was analyzed with Flowjo v10.1 (Flowjo, Ashland, OR).