Cd45 cd11b

The following antibodies were used to stain single cells: antibodies for DC staining - anti-mouse CD45 (30-F11, Invitrogen), anti-mouse CD11b (M1/70, TONBO), anti-mouse CD11c (N418, TONBO), and anti-mouse MHC II (AF6-120.1, eBioscience), anti-mouse CD115 (T38-320, BD), and anti-mouse Ly-6G (RB6–8C5, eBioscience). After incubation, the antibody/cell mixtures were left on ice for 30 minutes. The cells were then washed 3 times with 1 ml 2% FBS in PBS, and centrifuged at 300xg for 3 minutes. Cells were resuspended in 300 μ1 2% FBS in PBS, and separated with flow cytometry using the LSR II (BD Biosciences) for the CD45⁺CD11b⁺CD11c⁺MHCII⁺ DCs, CD45⁺CD11b⁺CD115⁺ macrophages, and CD45⁺CD11b⁺Ly-6G⁺ neutrophils. Results were analyzed with FACSDiva (BD Biosciences).

Cells from tumor, spleen, lymph nodes, and lung were mechanically dissociated, and the red blood cells were removed with ACK lysis buffer (Lonza, Allendale, NJ). Cells were blocked with Fc antibody and then labeled with various antibody combinations (Lin/CD45/CD31, Lin/CD45/Sca-1/CD44, CD45/CD11b) (BD Biosciences, San Jose, CA). Data were acquired with the BD LSRFortessa (Becton Dickinson) cell analyzer, and analysis was performed using Flowjo software. Cell sorting was performed using a Becton Dickinson cell sorter.