Granulocyte macrophage colony stimulating factor

Bone marrow-derived dendritic cells (BMDCs) were isolated from the bone marrow after 8 ~ 10 days of differentiation with granulocyte-macrophage colony-stimulating factor (20 ng/mL, Sino Biological) and IL-4 (10 ng/mL, Sino Biological). Macrophages were purified from tumor tissues with Anti-F4/80 MicroBeads (130-110-443, Miltenyi Biotech Inc.). BMDCs and macrophages were stained with the CFSE Cell Division Tracker Kit (423801, BioLegend). CT26-RFP cells were treated with drugs as indicated for 4 h and then added to the CFSE-labeled BMDC or macrophage culture at a 1:1 ratio. For flow cytometry, the cells were harvested and tested on a NovoCyte flow cytometer. BMDCs and macrophages that phagocytosed CT26 cells were CFSE and RFP-double-positive. For immunofluorescence, cells were fixed in 4% paraformaldehyde. Cell nuclei were stained with DAPI. The concentrations of IL-1β (EMC001b, Neobioscience), IL-12 (EMC006, Neobioscience), IL-18 (EMC011, Neobioscience), and CXCL9 (EK0733, BOSTER) in the supernatant after 24 h of co-culture were quantified using the ELISA Kit.