Cd45 percp 30f11

BMNC. PECs, lymph or splenic cells were washed in FACS buffer, blocked for 10 min in F_c block and incubated with various conjugated antibodies. To determine antigen specific T cells: CD3-FITC, OTI-CD8-dsRed, MHC I-Ova dextramer-APC (Immudex, Denmark and Tetramer core facility NIH, Bethesda, MD); to determine T cell activation: CD3-PerCp (145-2C11), CD4-PECy7(RM4-5), CD8-FITC (53-6.7), CD44-PE (IM-7), CD62L-APC (MEL-14); to determine monocyte populations: CD11c-APCCy7 (N418), CD11b-FITC (M1/70), Ly6C-PE (AL-21), Ly6G-APC (RB6-8C5), MHCII-PECy7 (M5/114.15.2), and CD45-PerCp (30F11), (eBioscience, San Diego, CA). Cells were analyzed using a BD FACSCanto II flowcytometer (BD Biosciences, San Jose, CA) and FlowJo analysis software v.8.7.3 (Treestar Software, Ashland, OR). Cell populations were determined by gating on CD4⁺, CD8⁺, (T cells) CD45^hi/CD11b⁺ (macrophages) and CD45^int/CD11b⁺ (microglia) Ly6CintLy6G⁺, (neutrophils) Ly6C⁺Ly6G⁻, (inflammatory monocytes) from a live cell gate (gating strategy as depicted in Supplementary Figs. 5, 7).

Single cells from tumours were isolated as described previously32 (link). Briefly, tumours were digested with 300 μg ml⁻¹ collagenase and 100 μg ml⁻¹ hyaluronidase (Stemcell Vancouver, BC, Canada), 0.25% trypsin (Mediatech, Corning, NY) and 0.1 mg ml⁻¹ DNase I (Worthington, Lakewood, NJ), filtered through 40-μm mesh and resuspended in HBSS with 2% FCS. Single cells were then stained with antibodies including the following: anti-mouse CD11c-Pac Blue (N418; 2.5 μg ml; Biolegend, San Diego, CA), anti-mouse F4/80-PE (BM8; 2 μg ml⁻¹), CD11b-FITC (M1/70; 2.5 μg ml⁻¹), CD45-PerCP (30-F11; 2 μg ml⁻¹), Ly6G-APC (1A8; 2.5 μg ml⁻¹) and Ly6C-PE-Cy7 (HK1.4; 2 μg ml⁻¹; Ebiosciences, CA). Cells were sorted with flow cytometry using an LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ) or an Accuri C6 flow cytometer (BD Biosciences). Analysis of flow cytometry data was done using FlowJo. CD11b⁺ myeloid cells were purified using CD11b-positive selection kit (STEMCELL). Some other antibodies include the following: CASP1 p10 (M20, 1 μg ml⁻¹) antibody (Santa Cruz Biotechnology, Santa Cruz, CA); CASP1 antibody (14F468, 1 μg ml⁻¹, Genetex, Irvine, CA); M2 Flag antibody (Sigma-Aldrich, St Louis, MO, 0.5 μg ml⁻¹); Casp11 antibody (17D9, 1 μg ml⁻¹, Novus Biologicals, Littleton, CO); Phos-JNK (G-7, 1 μg ml⁻¹) and JNK (D-2, 1 μg ml⁻¹) from Cell Signaling Technologies (Danvers, MA).