Diamidino-2-phenylindole (DAPI) (0.5 g/mL; Invitrogen, Grand Island, NY, USA). Isotype control antibodies were used as negative controls. Tissues were mounted, and the images were obtained by using a fluorescence microscope (Leica). For immunohistochemistry staining,Hu et al. Stem Cell Research Therapy 2015, 6:10 http://stemcellres.com/content/6/1/Page 5 ofthe quadriceps muscle was harvested at day 21 and fixed with paraformaldehyde and embedded in paraffin, and the sections were stained with hematoxylin and eosin and examined under a light microscope (Leica) by two pathologists who were blinded to the grouping conditions.Endothelial cell migration assayThe real-time cell analyzer (RTCA) migration assay and scratched wound assay were used to analyze the migration effect of iMSCs-Exo to HUVECs. The xCELLigence system (Roche Applied Sciences, Basel, Switzerland) used impedance as a readout and can continuously monitor the cellular responses of biologically active small-molecule compounds, producing time-dependent cellular response profiles. The electronic readout of cellsensor impedance is displayed in real-time as CI, a value directly influenced by cell attachment, spreading, or cell proliferation or a combination of these. The CI value at each time point is defined as Rn-Rb/Rb, where Rn is the cell-electrode impedance of the well with the cells and Rb is the background impedance of the well with only medium [35]. HUVECs (4 ?104 cells per well) were seeded into the upper chamber, and M200 containing 100 g/mL iMSCs-Exo or control medium was added into the lower chamber. The cells were incubated at 37 in 5 CO2 and monitored for 24 hours. For the scratched wound assay, 2 ?105 cells were seeded into 12-well plates and maintained at 37 to permit cell adhesion and the formation of a confluent monolayer. Next, these confluent monolayers were `scratch’-wounded by using the tip of a p200 pipet tip. The medium was removed and rinsed once with PBS to remove the debris and smooth the edge of the scratch and then replaced with fresh M200 + LSGS medium containing 100 g/mL iMSCs-Exo or control medium. Wound HS-173MedChemExpress HS-173 closure was monitored by collecting digital images at 0-, 12-, and 24-hour intervals after the scratch, and digital images were captured by using an inverted microscope (Leica). The PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28381880 images were obtained at the same position before and after incubation. The experiment was repeated three times. The level of wound closure was assessed by the ratio of closure area to initial wound (0 hours) as follows: Rn ? 0An??100 ; ABriefly, HUVECs were seeded at 5 ?104 cells/mL (100 L/well) in a 96-well plate. After quiescence for 12 hours, cells were treated with M200 + LSGS containing different doses of iMSCs-Exo (0, 12.5, 25, 50, and 100 g/mL) or control medium. At days 1, 2, 3, 4, and 5, CCK-8 solution (10 L) was added into medium and incubated for 3 hours at 37 . The amount of formazan dye generated by cellular dehydrogenase activity was measured for absorbance at 450 nm by using a microplate reader. The optical density values of each well represented the survival/proliferation of HUVECs. All of these experiments were performed in triplicate and repeated at least three times.Endothelial cell capillary-like tube formation assayIn vitro capillary-like tube formation was evaluated on growth factor-reduced Matrigel (BD Biosciences). At least 30 minutes before the experiment, 96-well plates were coated with Matrigel. Next, 2 ?104 HUVECs were seeded onto the pla.