Supplementary Figure 1.

Characterization of murine MSCs and MSC exosomes. (A) MSCs were characterized by flow cytometry analysis for surface markers (anti-CD105–PE, clone MJ7/18 [562759], anti-CD106–PE, clone 429 [561613], anti-CD44–APC, clone IM-7 [561862]; all BD Biosciences; anti-CD45–PE, clone 30-F11 [12-0451-82], anti-CD29–PECy7, clone HMb1-1 [25-0291-80], anti-stem cells antigen-1–APC, clone D7 [17-5981-81]; all eBioscience, Vienna, Austria) using the LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Inc, Ashland, OR). (B) MSC differentiation staining for adipocytes (lipid droplets) and osteoblasts (calcium deposit and alkaline phosphatase activity).⁵ (C) Western blot for exosomal markers flottilin-1 and alix. Western blot analysis was performed using 25 μg protein loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis⁸ and transferred to polyvinylidene difluoride membranes (0.45-μm pore size, 10401196; Whatman, Maidstone, UK). Ponceau staining (3504; Sigma-Aldrich) was used to confirm equal loading. Blots were incubated with primary antibodies to rabbit anti-alix, clone 3A9 (MCA2493; Bio-Rad Laboratories, Hercules, CA) and mouse anti–flotillin-1, clone EPR6041 (1333497; Abcam, Cambridge, UK). (D) Transmission electron microscopy images of exosomes isolated from MSCs. From a purified exosome suspension, 3 μL was put on a copper electron microscopy grid that was glow-discharged in air. By using an electron microscopy grid plunger freezer (Leica), excess suspension was removed by blotting for 1 second using filter paper (Whatman) at 95% humidity at room temperature and the grid was plunged into liquid ethane at -183°C. Samples were transferred to a Tecnai F20 Transmission Electron Microscope (ThermoFisher Scientific) using a Gatan cryo holder and images were recorded at 15,000× magnification on a Gatan 2kx2k CCD camera (Gatan, Pleasanton, CA) behind a 2001 energy filter operating at 20 electron-volts slit width. MW, molecular weight.