To enrich macrophages, mice were inoculated intraperitoneally with 2.5 mL of 2.98% Difo Fluid thioglycollate medium (BD, USA) for 3.5 days and macrophages were harvested from the peritoneal cavity. After washing twice with PBS, peritoneal macrophages were resuspended in RPMI 1640 medium with 10% fetal bovine serum (Biological Industries, Israel), diluted into 1.5 × 106 cells/mL, and cultured at 37°C/5% CO2 in 6-well plates at 4.5 × 106 cells/well, 12-well plates at 1.5 × 106 cells/well or 24-well plates at 5 × 105 cells/well. The cell purity was verified using flow cytometry through staining with APC anti-mouse/human CD11b (1:200, BioLegend, USA).
To determine whether GEVs could enter into mouse peritoneal macrophages, GEVs were stained using a PKH67 Green Fluorescent Cell Linker Kit (Sigma, St. Louis, MO, USA) [31 (link),32 (link)]. In detail, 50 μg of GEVs were dissolved in 100 μL of PBS and mixed with 1 mL Diluent C and 4 μL of PKH67 dye. The mixture was incubated for 4 min at room temperature (RT) in darkness and then added into 1 mL of 1% BSA to remove excess dye. The PKH67 labeled GEVs were washed with 5.5 mL PBS, ultracentrifuged at 100,000 × g for 1 h, and resuspended in 50 μL PBS.
To determine whether GEVs could enter into mouse peritoneal macrophages, GEVs were stained using a PKH67 Green Fluorescent Cell Linker Kit (Sigma, St. Louis, MO, USA) [31 (link),32 (link)]. In detail, 50 μg of GEVs were dissolved in 100 μL of PBS and mixed with 1 mL Diluent C and 4 μL of PKH67 dye. The mixture was incubated for 4 min at room temperature (RT) in darkness and then added into 1 mL of 1% BSA to remove excess dye. The PKH67 labeled GEVs were washed with 5.5 mL PBS, ultracentrifuged at 100,000 × g for 1 h, and resuspended in 50 μL PBS.