Dpbs without ca2 and mg2

Blood samples (~120 μL) were withdrawn from the facial vein of C57BL/6 (WT) recipient mice 8 weeks after transplantation with GFP⁺ bone marrow cells (GFP^+/-→WT) (n = 15) as well as of GFP^+/- (n =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.

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.