Granulocyte macrophage colony stimulating factor

Peritoneal macrophage (Mφ) isolation: Mice were sacrificed and the peritoneal liquid was collected. The pallet was diluted with RPMI1640 medium (Thermo Fisher, United States).
Bone marrow-derived Mφ (BMDM) culturing: Femurs from 4 weeks-age mice were collected and bone marrow was flushed out with cold PBS. Blood cell lysis buffer was added to the pellet for 5 min and the acquired cell medium was filtered by a 10 μm cell filter. After centrifugation, cells were dilated with complete solution (RPMI1640, 10% fetal borine serum, 1% Penicillin-Streptomycin solution, 50 ng/ml Granulocyte-macrophage colony-stimulating factor) (Stemcell technology, Canada). The medium was replaced every 2 days. On day 7, M0 Mφs were harvested for further experiments.
A total of 20 μl microparticles (Thermofisher, United States) diluted in 2000 μl 1% BSA were incubated at 37°C for 30 min and subsequent ultrasonic treatment for 5 min; then, 10⁵ Mφs were added to the microparticle solution and incubated for 1.5 h at 37°C. After centrifugation and washing, cells were diluted in 500 μl PBS and subjected to flow cytometry analysis at the fluoresceine isothiocyanate (FITC) wavelength (488 nm).

For peritoneal Mφ preparation, mice were anesthetized and sterilized PBS was injected into the abdominal cavity. After the massage, the liquids were isolated and centrifuged. The formula was diluted in a 1640 complete medium.
For the bone marrow-derived macrophage (BMDM), 4 week-old male mice were sacrificed and bone marrow from femurs was incubated with blood cell lysis buffer (Beyotime, China) for 5 minutes. Filtered cells were cultured with the 1640 medium in the presence of the granulocyte-macrophagecolony-stimulating factor (STEMCELL Technologies, Canada) at a concentration of 55 ng/ml. Mφs were collected on day 7.
10⁵ Mφs were cultured with prepared microparticle solution (Thermo Fisher, USA) for 2 hours at 37°C and then mounted at the fluorescein isothiocyanate (FITC) wavelength (488 nm).