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).
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).