Cd11b pe cy7 clone m1 70

Leukocytes from the brain were isolated using a standard protocol. Briefly, the brain was collected in RPMI 1640 with 3% (v/v) FCS and mechanically dissociated. A 30% Percoll/brain homogenate suspension was underlain with 70% Percoll in PBS and then centrifuged at 1050×g for 25 min. Cells in the interphase were collected for further analyses. Expression of MHC-II by microglia was determined by flow cytometry using the antibodies CD45.2-FITC (clone 104, dilution 1:100), CD11b-PE-Cy7 (clone M1/70, dilution 1:400), and MHC-II-APC (clone M5/114.15.2, dilution 1:200) purchased from Thermo Fisher. Microglia were gated as live CD45^intCD11b⁺ cells.

Splenocytes were prepared as described before. Monocytes/macrophages were enriched by a pre-isolation step using NKp46, CD3e and CD19 MACS beads negative selection (all Miltenyi Biotech, Germany). Subsequently, cells were stained for CD3e PerCP-eFluor 710 (clone 17A2, Thermo Fisher Scientific), CD19 FITC (clone 1D3, BD Biosciences), CD11c APC (HL3, BD Biosciences), CD11b PE-Cy7 (clone M1/70, Thermo Fisher Scientific) and HLA-DR BV605 (clone G46-6, BD Biosciences) and sorted using a BD FACS Aria III (BD Biosciences). Nonspecific binding through Fc gamma receptors was blocked by a mixture of anti-CD16 and anti-CD32 antibodies (Fc Block; BD Biosciences). Post sort analysis using FlowJo software (Version 10.8.1; BD Biosciences) showed an average purity of 94.4% for splenic CD11b+ monocytes/macrophages.

Blood samples (~120 μL) were withdrawn from the facial vein of C57BL/6 (WT) recipient mice 8 weeks after transplantation with GFP⁺ bone marrow cells (GFP^+/-→WT) (n = 15) as well as of GFP^+/- (n = 9) and WT (n = 6) animals. Samples were quickly collected in EDTA-coated tubes (Starstedt, Montreal, Quebec, Canada) to prevent coagulation. A volume of 35 μL of DPBS without Ca²⁺ and Mg²⁺ (Sigma-Aldrich, St Louis, MO) was added to 65 μL of blood and incubated for 20 min on ice with purified rat anti-mouse CD16/CD32 antibody diluted 1:100 (clone 2.4G2; BD Biosciences) to block non-specific binding of IgGs to Fc receptors. Samples were washed and resuspended in 100 μL of DPBS after being centrifuged at 300 x g for 10 min. Cell suspensions were then labeled with the following rat anti-mouse antibodies for 40 min at 4°C: PE-Cy5-CD45 (clone 30-F11; BD Biosciences), APC-CD115 (clone AF598; eBioscience, San Diego, CA), PE-Cy7-CD11b (clone M1/70; eBioscience), V450-Ly6C (clone AL21; BD Biosciences) and PE-Ly6G (clone 1A8; BD Pharmingen, San Jose, CA). Red blood cells were lysed with BD Pharm Lyse™ (BD Biosciences) during 30 min at room temperature, and the recovered leukocytes were washed and resuspended in DPBS for analysis. Flow cytometry analysis and data acquisition were performed using a BD SORP LSR II and the BD FACSDiva software, respectively.

If not indicated otherwise, all chemicals were obtained from Sigma-Aldrich Chemie GmbH. The following antibodies were applied for Western blot analyses: polyclonal anti-NOS (pan) (Cell Signaling Technology, Leiden, The Netherlands) and anti-β-actin (clone AC-15) and horseradish peroxidase-labeled polyclonal rabbit anti-mouse and polyclonal goat anti-rabbit antibodies (Dako, Hamburg, Germany).
The following antibodies were applied for flow cytometric analysis: PE-CD80 (clone 16-10A1) and APC-Cy7-CD11b (clone M1/70) from BD Bioscience (Heidelberg, Germany); APC-MHCII (clone M5/114.15.2), PE-Cy7-Gr1 (clone RB6-8C5), Percp-Cy5.5-CD11c (clone N418) and PE-Cy7-CD11b (clone M1/70) are from eBioscience (Frankfurt, Germany); Percp-Cy5.5-CD4 (clone GK1.5), APC-CD4 (clone GK1.5) are from Biolegend GmbH (Fell, Germany). BODIPY was obtained from Life Technologies (Carlsbad, CA, USA).

Prior to (day 0) and on days 4, 6, 8 and 10 following infection, blood samples (~120 μL) were drawn from the facial vein of CCR2^-/-→WT and WT→CCR2^-/- chimeric mice as well as age- and sex-matched WT and CCR2^-/- controls (n = 5 mice per group). Samples were quickly collected in EDTA-coated tubes (Starstedt, Montreal, Quebec, Canada) to prevent coagulation. A volume of 35 μL of DPBS without Ca²⁺ and Mg²⁺ (Sigma-Aldrich) was added to 65 μL of blood and incubated for 20 min on ice with purified rat anti-mouse CD16/CD32 antibody diluted 1:100 (clone 2.4G2; BD Biosciences) to block non-specific binding of IgGs to Fc receptors. Samples were washed and resuspended in 100 μL of DPBS after being centrifuged at 300 x g for 10 min. Cell suspensions were then labeled with the following rat anti-mouse antibodies for 40 min at 4°C: PE-Cy5-CD45 (clone 30-F11; BD Biosciences), APC-CD115 (clone AF598; eBioscience, San Diego, CA), PE-Cy7-CD11b (clone M1/70; eBioscience), V450-Ly6C (clone AL21; BD Biosciences) and PE-Ly6G (clone 1A8; BD Pharmingen, San Jose, CA). Red blood cells were lysed with BD Pharm Lyse™ (BD Biosciences) for 30 min at room temperature, and the recovered leukocytes were washed and resuspended in DPBS. Flow cytometry analyses and data acquisition were performed using a BD SORP LSR II and the BD FACSDiva software, respectively.