Fitc rat anti mouse cd11b

A single-cell suspension was prepared from BM following aspiration from the femur and tibia with a 26-gauge syringe and PBS. A single-cell suspension was prepared from spleen following mechanical dissociation and filtration through a 40-µm cell filtration cap. Single-cell suspensions were washed in staining buffer (0.2% BSA and 5 mM glucose in PBS), and 2 × 10⁶ cells per sample were resuspended in the staining buffer for staining with the following anti-mouse antibodies. Lineage exclusion cocktail (all antibodies used at 1:400): FITC rat anti-mouse CD41 (BioLegend; 133904), FITC rat anti-mouse CD45R/B220 (BioLegend; 103206), FITC rat anti-mouse CD3 (BioLegend; 10024), FITC rat anti-mouse CD11b (BioLegend; 101206), and FITC rat anti-mouse Gr-1 (BioLegend; 108406). Erythroid markers: FITC rat anti-mouse Ter119 1:200 (BioLegend; 116206), Pacific Blue rat anti-mouse Ter119 1:200 (BioLegend; 116232), biotin rat anti-mouse Ter119 1:200 (BioLegend; 116203), PE rat anti-mouse CD71 PE 1:200 (eBioscience; clone R17217), and DRAQ5 1:500 (eBioscience; 65–0880). Secondary antibody: strepavidin-APC-Cy7 (BioLegend;, 405208). FACS was performed on a MACS Quant Analyzer (Miltenyi), and FACS analysis was completed on FlowJo version 10.

For cell painting analysis, 10⁶ Ras cells were washed and stained in PBS 0.05% BSA with CMRA (orange, for p65^+/+Ras cells) or CFSE (green, for p65^−/−Ras cells) fluorescence dyes according to the manufacturer (Invitrogen). Stained cells were then mixed in a 1:1 ratio and applied to 35 mm dishes containing coverslips. Two hours later, isolated MΦs were applied and incubated at 37°C with 5% CO₂ for an additional 16–20 hr. Cells were fixed with 4% paraformaldehyde in PBS and mounted onto slides with DAPI-containing mounting solution (Electron Microscopy Sciences). Mounted cells were then observed with a fluorescence microscope and red to green cell ratios were calculated. For apoptosis analysis, cells were coincubated with harvested MΦs overnight. The next day, cells were harvested by trypsinization and stained with FITC-rat anti-mouse CD11b (BioLegend). Then cells were washed with Annexin V staining buffer and stained for Annexin V and 7-AAD with an phycoerythrin (PE) conjugate Annexin V staining kit (BD Pharmingen) as recommended by the manufacturer and analyzed by FACS. For CTL assays, p65^+/+Ras or p65^−/−Ras cell-specific CTLs were prepared (Supplemental Experimental Procedures) and cocultured with tumor cells in 96 well plates with 5–10 units/ml of IL-2. Thirty-six to 48 hours later, plates were trypsinized and viable cells were counted by a trypan blue exclusion assay.