Pe cy7 anti cd11b clone m1 70

Single cell suspensions deriving from brain and spleen tissue were incubated for 30 min at 4°C in Zombie Aqua live/dead exclusion dye (1:1000 from stock in HBSS (without Mg²⁺ and Ca²⁺), LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit, Molecular Probes, Thermo Scientific). Next, cells were washed in FACS buffer (2% (v/v) FBS (heat-inactivated), 5 mM EDTA, 0.01% (v/v) NaN₃) and resuspended in Fc receptor blocking antibodies (1:20 from stock in FACS buffer, anti-mouse CD16/32 TruStain fcX, Biolegend). After 5 min incubation, cells were treated with antibody master mix for surface marker staining and incubated for 15 min at 4°C, followed by a last wash in FACS buffer. For myeloid cells (microglia, dendritic cells, macrophages) we used the following fluorophore-conjugated antibodies against surface markers: PE-Cy5.5 anti-CD45 (clone 30-F11, eBioscience, Thermo Scientific), PE-Cy7 anti-CD11b (clone M1/70, eBioscience, Thermo Scientific), APC anti-CD11c (clone N418, BioLegend), Alexa Fluor 488 anti-MHC class II (clone M5/114.15.2, Biolegend).

Mice were sacrificed, and bronchoalveolar lavage (BAL) cells were collected by flushing the exposed trachea after cannulation with an 18-gauge, ½-in. cannula connected to a 1-ml syringe. The BAL cells were harvested by flushing the lungs with 10 % 1XRPMI and centrifugation. The cells were resuspended for flow cytometric analysis in appropriate stains. They were first blocked with anti-CD16/32 (FcR block, eBiosciences) to prevent non-specific staining and then stained with APC Cy7-anti-F4/80 (clone BM8, Biolegend), PE Cy7-anti-CD11b (clone M1/70, eBioscience), APC-anti-CD11c (clone N418, Biolegend), and FITC-anti-Gr1 (clone RB6-8C5, BD Pharmingen) and gated according to Hall et al. [22 (link)]. Briefly, cells were first gated as F4/80⁺ or F4/80⁻ cells. F4/80⁺ cells that are CD11b^high were referred to as macrophages and those that are CD11c^high/CD11b^low as alveolar macrophages. DCs were gated as F4/80^low CD11c^high and monocytes as F4/80^low CD11c^lowCD11b^mid. Finally, neutrophils were gated as F4/80^low CD11c^lowCD11b^high and Gr1^high. Data were collected using the LSRII flow cytometer and analyzed with FACSDiva 6.1 software (BD Biosciences).

Microglia were isolated as described previously (Galatro, Vainchtein, Brouwer, Boddeke, & Eggen, 2017b). Briefly, mice were perfused using PBS (Lonza, BE17‐512F) and brain and spinal cord were isolated in HBSS (Gibco, 14170‐088) containing 15 mM HEPES (Lonza, BE17‐737E) and 0.6% glucose (Sigma‐Aldrich, G8769) (=Medium A). Tissue was mechanically disrupted to obtain a single cell suspension and myelin was removed using 24.4% percoll (GE Healthcare, 17‐0891‐01) density gradient centrifugation at 950g. Cells were incubated 15 min in anti‐Cd16/32 blocking buffer (clone 93, eBioscience, 14‐0161‐85) and stained with anti‐Cd11b‐PE‐Cy7 (clone M1/70, eBioscience, 25‐0112‐81), anti‐Cd45‐FITC (clone 30‐F11, eBioscience, 11‐0451‐85), and anti‐Ly6c‐APC (clone HK1.4, Biolegend, 128016) antibodies 30 min on 4°C. Cells were sorted on a MoFlo XDP Cell Sorter (Beckman Coulter) in RNAlater (Qiagen, 76104) in siliconized tubes. Sorted cells were centrifuged at 5,000g (RNAlater) and lysed in RLT+ lysis buffer (Qiagen, 74034).