F4 80 efluor450 clone bm8

Six days post-infection, mice were exsanguinated and perfused as above; brains were harvested and homogenized in RPMI media-containing 0.5 mg/mL collagenase (Gibco LifeTechnologies), 0.01 mg/mL DNAse I (Roche) and 2 mM EDTA. Infiltrating cells were separated on a 33.3 % Percoll solution. Cells were stained for flow cytometry analyses with the following; Zombie Aqua Dye-V500 (BioLegend), anti-CD45-APC-eFluor780 (clone 30-F11, eBioscience), anti-CD4-PerCP Cyanine5.5 (clone RM4-5, eBioscience), anti-CD8alpha-PE (clone 53-6.7, eBioscience), anti-TCRbeta-FITC (clone H57-597, eBioscience), anti-CD19-BV421 (clone 6D5, BioLegend), F4/80-eFluor450 (clone BM8, eBioscience), anti-CD11b-APC (clone M1/70, eBioscience), anti-CD11c-PerCP Cyanine5.5 (clone N418, eBioscience), anti-Ly6C-PE (clone HK1.4, eBioscience), and anti-Ly6G-FITC (clone 1A8, BioLegend). Leukocytes were gated as CD45^hi cells.

Freshly prepared BMDM were used for each experiment. Femur and tibia were aseptically removed from mice, the bone ends were cut off, and the bone marrow cells were flushed out using PBS (4–8°C; Gibco, Darmstadt, Germany). The cell suspension was washed twice with PBS by centrifugation (490 × g, 5 min) and cultured in Petri dishes (ø 85 mm, Sarstedt, Numbrecht, Germany) in Dulbecco’s modified Eagle’s medium with high glucose and l-glutamine (DMEM) supplemented with 10% FCS, 1% non-essential amino acids (all from PAA, Coelbe, Germany), 1% glutamine (Biochrom, Berlin, Germany), 50 μM 2-­mercaptoethanol (Gibco, Darmstadt, Germany), and 20 ng/ml macrophage colony-stimulating factor (M-CSF) (PeproTech, Hamburg, Germany) for 3 days. Medium was changed on day 3 and non-adherent cells were removed by washing with PBS. Adherent BMDM were harvested at day 6 and used for experiments. The purity of the cultured macrophages ranged from 95 to 99%, as determined by flow cytometry using the macrophage-specific markers CD11b and F4/80 (F4/80 eFluor450 (clone: BM8)) and CD11b APC (clone: M1/70; both eBioscience, Frankfurt, Germany).