Ly6g apc

Active caspase-1 activity in monocytes was determined using the FAM FLICA Casp-1 assay kit (Immunochemistry Technologies, Davis, CA, USA) (Table S1), following the manufacturer’s instructions. In a subset of bone-marrow-transplanted animals, blood from Tg26^+/−/ApoE^−/−/Casp-1^+/+ (n = 4) and Tg26^+/−/ApoE^−/−/Casp-1^−/− mice (n = 4) was collected via tail bleeding. After removal of red blood cells, peripheral blood mononuclear cells (PBMCs) were incubated with monocyte markers, including CD11b-PE-Cy7, Ly6C-Flour450 (eBioscience, San Diego, CA), and Ly6G-APC (Bio legend, San Diego, CA, USA), for 30 min at room temperature (Table S1). After washing with 1xPBS, the cells were then stained with caspase-1 inhibitor FAM-YVAD-FMK at 37 °C for 1 h, followed by washing with the 1x apoptosis wash buffer provided in the kit. In the end, the supernatants were discarded, and the cells were resuspended with 300 μL of 1× apoptosis wash buffer. Caspase-1 activity in monocytes was recorded using BD LSR Fortessa flow (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). The analysis and histogram graphics were performed using the FlowJo software (version FlowJo_v10.9.0_CL) (Ashland, OR, USA).

Cells harvested from the spleen were used for single color and multi-color staining controls. Control and tumor samples were resuspended at a concentration of 10⁶ cells/100 uL in FWB in round bottom polystyrene tubes. Cells were stained with the following antibody fluorophore conjugates: CD45-PerCP/Cy5.5, CD11c-Pacific Blue, CD11b-AF700, MHCII-APC/Cy7, Ly6c-BV510, Ly6g-APC, CD38-FITC (Biolegend, San Diego, CA), F480-PE/Cy7 (Tonbo Bioscences, San Diego, CA), CD206-PE (R&D Systems, Minneapolis, MN) and propidium iodide (PI, Enzo Life Sciences, Farmingdale, NY). All antibody staining took place for 30 min at 4 °C in the dark. For all experiments, cells were acquired on the BD LSRII Fortessa flow cytometer. Compensation and sequential gating was performed with FlowJo software (FlowJo LLC, Ashland, OR). Populations of myeloid cells that were identified included macrophages, dendritic cells, granulocytic myeloid derived suppressor cells (G-MDSC), monocytic myeloid derived suppressor cells (M-MDSC) and M0, M1 and M2 macrophage phenotypes (Supplementary Fig. S1).