Af2724

Isolated or immunocaptured exosomes were tested for the presence of CD63, CD81, CD34, GAPDH, CD200, CD44 and CD105 using western blots as previously described [14] (link). Briefly, 10 µg of exosomes were lysed with Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA), separated on 7–15% SDS/PAGE gels and transferred onto PVDF membrane (Millipore, Billerica, MA, USA) for western blot analysis. Membranes were incubated overnight at 4°C with antibodies specific for: CD63 (1∶200, sc-15363, Santa Cruz, CA, USA), CD34 (1∶2000, ab81289, Abcam, Cambridge, MA, USA), CD81 (1∶200, PA5-13582, Thermo Fisher, Pittsburgh, PA, USA), GAPDH (1∶500, FL-335, Santa Cruz, CA, USA), CD200 (1∶2000, AF2724, R&D, Minneapolis, MN, USA), CD44 (1∶1000, ab41478, Abcam, Cambridge, MA, USA), CD105 (1∶1000, ab169545, Abcam, Cambridge, MA, USA), platelet IIb/IIIa (1∶200, sc-73544, Santa Cruz, CA, USA). Next, the HRP-conjugated secondary antibody (1∶5000, Pierce, Thermo Fisher, Pittsburgh, PA, USA) was added for 1 hr at room temperature (RT) and blots were developed with ECL detection reagents (GE Healthcare Biosciences, Pittsburgh, PA, USA). The intensities of the bands on exposed films were quantified using Image J software (NIH, USA).

For flow cytometry analysis, followed by dissociation of the expanded MSCs, 1 × 10⁵ cells were suspended in 100 μl of 2% FBS in PBS and incubated with primary antibody (diluted 200×) (CD45-PE-CyTM7 (368510, Biolegend, San Diego, CA, USA); CD90-APC (328114, Biolegend, San Diego, CA, USA); CD29-APC (17-0299-42, Invitrogen, USA); CD105-PE (85-12-1057-42, eBioscience, San Diego, CA, USA); and CD200 (0.25 μg/10⁶ cells, AF2724, R&D Systems, USA); Msx1 (diluted 200×, NBP2-57969, NOVUS, USA).) for 1 h on ice. The cells were then washed and suspended in 100 μl of 2% FBS in PBS containing secondary antibody (diluted 1000×) for 30 min on ice. Fluorescence was detected using a CytoFLEX S flow cytometer (Beckman Coulter) and data were analyzed using the FlowJo software. Gating strategy used for analyses is available in Supplementary Fig. 14.