Fitc conjugated cd45

FACS was performed using BD FACSAria III (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). To analyze the mesenchymal stem cell markers, 10⁵ cells were stained with antibodies, particularly PE-conjugated CD105 (Bio-Rad, Hercules, CA, USA, MCA1557,), PE-conjugated CD90 (BioLegend, San Diego, CA, USA, 555596), PE-conjugated CD73 (BioLegend, 344004), FITC-conjugated CD45 (BioLegend, 555482), FITC-conjugated CD34 (BioLegend, 343504), and PE-conjugated CD14 (Bio-Rad, MCA1568). To determine the EV-specific marker, isolated EVs were incubated with 4% w/v aldehyde/sulfate-latex beads (Thermo Fisher Scientific, Rockford, IL, U.S, A37304) at room temperature overnight on rotation. Bead-binding extracellular cells were centrifugated at 1800× g for 10 min and washed with 500 µL of PBS. The pellet was resuspended with 50 µL of PBS containing CD63 (BioLegend, 353003) and CD81 (BioLegend, 349505) at 4 °C for 1 h. All antibodies were conjugated with PE fluorescence dye. Samples were washed with 500 µL of PBS and centrifuged at 1800× g for 10 min. The pellet was resuspended with PBS. Gating of exosome-decorated beads approximately 4 µm in diameter was analyzed using BD FACSAria III (BD Biosciences, San Jose, CA, USA). At least 10,000 events were acquired on flow cytometry. Data analyses was performed using BD FACSDiva™ software version 6.1.3 (BD Biosciences, San Jose, CA, USA).

One million iMSCs and UCB-MSCs were stained with PE-conjugated CD73, PE-conjugated CD105, PE-conjugated CD90 (Miltenyi Biotec, Somerville, MA, USA), PE-conjugated CD34, and FITC-conjugated CD45 (BioLegend, San Diego, CA, USA) for 30 min at 4 °C. Stained cells were washed twice in PBS, and FACS was performed with a BD Accuri C6 flow cytometer (BD Biosciences, San Diego, CA, USA). The antibodies used are listed in Supplementary Table S6.