Permwash staining kit

Tumor lysates were prepared by mincing the tumor in DMEM (Sigma) and incubating in collagenase (Roche) at 37°C for 1 h. After washing in PBS, the cells were filtered through 70‐μm filters (BD Biosciences). 5 × 10 cells were re‐suspended in HBSS (Hank's balanced salt solution, Lonza) supplemented with 0.5% BSA (Sigma). Staining was performed at 4°C for 20 min, with the following antibodies: anti‐mouse CD45 (clone 30‐F11, Biolegend San Diego, CA, USA), anti‐mouse F4/80 (clone BM8, Biolegend San Diego, CA, USA), anti‐mouse cd11c (clone N418, Biolegend San Diego, CA), anti‐mouse I‐A/I‐E (clone M5/114.15.2, Biolegend San Diego, CA, USA); anti‐mouse TNFα (clone MP6‐XT22, BD Pharmingen), anti‐mouse Ly6C (clone HK1.4, eBioscience; San Diego, CA, USA); anti‐mouse NOS2 (clone 6/iNOS/NOS Type II, BD biosciences, San Diego, CA, USA) and mouse IgG2a K (BD biosciences, San Diego, CA, USA). For intracellular staining, Cytofix/Cytoperm and Permwash staining kit (BD Pharmingen) were used. Cells were detected using the BD FACS Canto II cytofluorimeter and analyzed with FlowJo software.

Preparation and staining from BM and splenic cells were done as described^{24 (link)}. Briefly, cells from BM were isolated by flushing the femur of the indicated mice with PBS. ACK lysis was applied for 1 min and cells were washed three times with PBS 1× supplemented with 10% FBS followed by staining with the indicated antibodies and flow cytometry. Single cells were isolated from spleen by mashing on 70 μm filters followed by ACK lysis for 2 min and washing with PBS 1× supplemented with 10% FBS. Single-cell suspensions were plated in 96-well plates. We performed surface staining at 4 °C for 15 min with the flow antibodies listed in Supplementary Table 4. For intracellular staining, Cytofix/Cytoperm and Permwash staining kit (BD Pharmingen) were used according to the manufacturer’s instructions. We performed intracellular staining 4 °C for 30 min with flow antibodies listed in Supplementary Table 4.