Hoechst 33258

Cell apoptosis assay was assessed by using PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, CA, USA) and Hoechst 33258 staining as previously described [18]. Briefly, following different treatments, MC3T3-E1 cells were collected and resuspended in 100 μL binding buffer, stained with Annexin V-PE and 7-AAD at room temperature in the dark for 15 min. Stained cells were analysed using a FACSCantoⅡ flow cytometer (BD Biosciences, USA). For pathway blocking experiments, cells were pre-incubated with PD98059 (10 μM) for 2 h, then cells were treated with EPC-MVs or EPC-MVs-miR126. Cell apoptosis rate was quantified in three times independent experiments.
For Hoechst 33258 staining, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min after EPC-MVs or EPC-MVs-miR126 treatment for 48 h, then stained with Hoechst 33258 (Sigma, Louis, MO, USA) according to the manufacturer’s instruction. Morphologic changes were observed under a fluorescence microscopy in four independent fields.

Cell apoptosis was first analyzed using a fluorescein isothiocyanate (FITC)-Annexin V apoptosis kit (BD Biosciences, San Jose, CA, USA). In brief, the C6661 and 6-10B cells were resuspended in 1 × Annexin V binding buffer supplemented with 10 mM HEPES/NaOH (pH = 7.4) (1 × 10⁶ cells/mL). The cells were cultured with FITC-Annexin V (5 µL) and propidium iodide (5 µL) at 37 °C for 20 minutes and analyzed using a flow cytometer (BD Biosciences).
Apoptosis of cells was further examined using Hoechst 33,258 staining. In short, the cells were seeded in 6-well plates and cultured in medium at 37 °C for 24 hours. Then, the cells were trypsinized and centrifuged at 1000 rpm for 5 minutes. After PBS washes, the cells were stained with 300 µL Hoechst 33,258 (C1017, Beyotime) at room temperature for 5 minutes and then observed under the fluorescence microscope at a wavelength of 365 nm. Three random fields were observed at a × 20 magnification for cell apoptosis detection.

To determine the purification efficiency of MACsorted CECs, flow cytometry of cell isolations was performed directly after MACS. Therefore about 1x10⁶ cells from (1) single cell suspensions directly after enzymatic digestion without MACS (pre-sort fraction), (2) single cell suspension after MACS from flow through (post-sort negative-fraction) and (3) single cell suspension after MACS adherent to the MACS column (post-sort positive-fraction) were taken, centrifuged at 300 g for 10 min at 4°C and resuspended in ice cold fluorescence-activated cell sorting-(FACS)-buffer consisting of 2 mM EDTA and 1 % FCS in PBS. To prevent unspecific binding of antibodies, all samples were incubated with mouse Fc block (BD Bioscience) for 5 min. Afterwards all samples were labeled with anti-CD31-PE antibody (1:1000, BD Bioscience) for 30 min at 8°C. To exclude dead cells from analysis, the samples were incubated with 1 μg ml^-1 Hoechst-33258 (BD Pharmingen) shortly before flow cytometry analysis. Isotype controls were generated to exclude unspecific binding. Finally, all samples were analyzed using a CyFlow cytometer (Partec, Goerlitz, Germany) in combination with FlowJo software (Tree Star Inc., Ashland, USA). For embryonic CEC injections into hearts with cryoinfarctions, only preparations with > 85 % purity of CECs were used.

Flow cytometry and CSC gating were performed as previously described [28 ]. Briefly, cells were stained with anti-CD44-APC (559942; BD Biosciences, Franklin Lakes, NJ), anti-CD24-PE (555428; BD Biosciences), anti-EpCAM-PerCP-Cy5.5 (347199; BD Biosciences) antibodies, Hoechst 33258 (BD Biosciences), propidium iodide (PI; 556463; BD Biosciences), and Annexin V (Biolegend, San Diego, California; 640918). Flow cytometric analysis was performed on single cell suspensions for the CD44^hi/CD24^lo/EpCAM⁺ CSC phenotype.