Co-culture of BMMSCs and macrophages

To prevent the release of exosomes from BMMSCs, BMMSCs were treated with exosome inhibitor GW4869. In brief, BMMSCs were seeded in a six-well plate at the density of 1 × 10⁶ cells/well and treated with 10% GW4869 (D1692-5MG, Sigma-Aldrich, St. Louis, MO, USA). Cells and supernatant were collected after 24-h treatment and used for further analyses. Whether exosome secretion in BMMSCs treated with inhibitor GW4869 was occluded or not was ascertained by observation under an electron microscope.

Thereafter, BMMSCs were inoculated in the basolateral chamber of a 24-well transwell at 1 × 10⁴ cells/well, while macrophages were seeded in the apical chamber. After 24-h co-culture, macrophages were collected. The expression of miR-124-3p and Ern1 was determined. BMMSCs were transfected with GW4869 (at the final concentration of 10 nM), or macrophages treated with miR-124-3p mimic, miR-124-3p inhibitor, or their relevant controls (at the final concentration of 50 nM).

Next, exosomes extracted from these BMMSCs were labeled by PKH67 (Green) staining solution (MINI67-1KT, Sigma-Aldrich, St. Louis, MO, USA) and co-cultured with macrophages for 48 h. The exosomes at a concentration of 100 μg/mL were adopted to treat cells in vitro. Thereafter, the expression of miR-124-3p, Ern1, Arg1, Ym1, and Fizz was determined by RT-qPCR and western blot analysis.