Ly 6g apc 1a8

C57BL/6J mice aged 6–8 weeks were intraperitoneally injected with 700 mg of alum and evaluated 12 h later. The peritoneal cavity was washed with 5 ml of PBS. Peritoneal exudate cells (PECs) were analyzed by flow cytometry. The recruitment of neutrophils was visualized with CD11b-PE (M1/70; BD Biosciences) and Ly-6G-APC (1A8; eBioscience). PI (Sigma) was used to exclude dead cells. Neutrophil (CD11b⁺Ly6G⁺) recruitment was analyzed on a CytoFLEX (Beckman Coulter) (45 (link)).

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).