Cd49e pe

The immune phenotype of EVs was analyzed using flow cytometry (BD FACS Aria III, BD Bioscience, East Rutherford, NJ, USA) by immunostaining with the CD49e-PE (Sony, Tokyo, Japan), CD63-FITC (Biolegend, San Diego, CA, USA), Sca1-APC/Cy7 (BioLegend), CD45-PE/Cy7 (BioLegend), CD9-APC (Biolegend) and CD44-APC/Cy7 (BioLegend) antibodies.

MSCs were isolated from subcutaneous adipose tissue of the adult mice (8–9 weeks old). Adipose tissue were dissected into small pieces and digested with 0.2% collagenase II (Dia-M, Russia) in a shaker-incubator at 37 °C, 120 rpm for one hour. Suspended cells were pelleted (500 g for 5 min), washed once in PBS (PanEco, Russia) and re-suspended in DMEM (PanEco, Russia) supplemented with 10% fetal bovine serum (Gibco, UK) and 2 mM l-glutamine (PanEco, Russia). MSCs were maintained at 37 °C, 5% CO₂ with culture medium being replaced every 3 days. MSCs from passages 3 and 4 were used for the experiments.
MSCs were differentiated into three lineages: adipogenic, chondrogenic and osteogenic and it was confirmed by detection of lipid droplets (Oil Red dye staining), glycosaminoglycans and mucins (1% alcian blue staining) and calcium deposits (5% AgNO₃ staining), respectively. Immune phenotype was determined using monoclonal antibodies to CD90.2-PerCP (1301575; Sony, USA), CD44-APC/Cy7 (103028; BioLegend, USA), CD73-Alexa Fluor647 (127208; BioLegend, USA), CD49e-PE (1119525; Sony, USA), Sca1-APC/Cy7(108126; BioLegend, USA), CD29-PE (102208; BioLegend, USA), CD11b-PE/Cy7 (101216; BioLegend, USA), CD10-PE (312203; BioLegend, USA), CD45-PE/Cy7 (103114; BioLegend, USA). Expression of CD markers was analyzed by flow cytometry (BD FACS Aria III (BD Bioscience, USA)).

The immune phenotype of CIMVs-MSCs and MSCs-derived EVs was characterized by immunostaining with the following monoclonal antibodies: Sca1-APC/Cy7 (BioLegend, USA), CD49e-PE (1119525; Sony, USA), CD44-APC/Cy7 (BioLegend, USA), CD45-PE/Cy7 (BioLegend, USA), CD9-APC (Biolegend, USA), CD63-FITC (Biolegend, USA). CIMVs and EVs were analyzed by flow cytometry (BD FACS Aria III. BD Bioscience, USA), the 405 nm laser was used for better distinguish CIMVs and and EVs from debris.