Preosteoblast‐derived exosomes were tagged with PKH67 (PKH67 Green Fluorescent Cell Linker kit, (Sigma). MC4exo were mixed with diluted PKH67 and incubated at 37°C for 5 min.
18 (link) Following incubation, exosome‐free medium containing FBS (10%) was added to stop the reaction. BMMΦ were seeded onto glass cover slips and placed in 6‐well plates. Tagged‐exosomes were added (1000 particle/cell). After 2, 24 and 48 h, cells were fixed with 4% paraformaldehyde (PFA) for 30 min and permeabilized with 0.5% Triton X‐100 in PBS for 10 min. Cells were then incubated with Phalloidin‐AlexaFluor660 (ThermoFisher) for 30 min and nuclei were stained with DAPI (ThermoFisher). Fluorescence was observed using the Leica THUNDER imaging system (Leica Microsystem). MC4exo uptake was confirmed via z‐stack imaging taken at 63x magnification.
18 (link) Following incubation, exosome‐free medium containing FBS (10%) was added to stop the reaction. BMMΦ were seeded onto glass cover slips and placed in 6‐well plates. Tagged‐exosomes were added (1000 particle/cell). After 2, 24 and 48 h, cells were fixed with 4% paraformaldehyde (PFA) for 30 min and permeabilized with 0.5% Triton X‐100 in PBS for 10 min. Cells were then incubated with Phalloidin‐AlexaFluor660 (ThermoFisher) for 30 min and nuclei were stained with DAPI (ThermoFisher). Fluorescence was observed using the Leica THUNDER imaging system (Leica Microsystem). MC4exo uptake was confirmed via z‐stack imaging taken at 63x magnification.
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