Fig. 2

(HR + P)-EMVs showed less effects on inducing oxidative stress and apoptosis compared with HR-EMVs in hypoxia-reoxygenated cardiomyocytes. HR-injured AC16 cells were co-cultured with different concentrations (10^7, 10^8 and 10^9 particles/ml) of HR-EMVs for 24 h: (A) CCK-8 assay for AC16 cell viability. (B) Flow cytometric detection of AC16 cell apoptosis by Annexin V-FITC/PI double staining. (C) Quantitative analysis of apoptotic cells. Cells of annexin V+/PI − or annexin V+/PI + were added together to calculate the percentage of apoptotic cells. HR-injured AC16 cells were co-cultured with 10^9 particles/ml of HR-EMVs, (HR + D)-EMVs and (HR + P)-EMVs for 24 h: (D) CCK-8 assay for AC16 cell viability. (E) LDH assay for lactate dehydrogenase activity in the cell supernatant. (F) Flow cytometry detection of reactive oxygen species (ROS) levels in AC16 cells after DCFH-DA staining. (G) Quantitative analysis of ROS. (H) Flow cytometry detection of apoptotic rate in AC16 cells after Annexin V-FITC/PI double staining. (I) Quantitative analysis of apoptotic cells. (J-K) Western blotting analysis of Bax, Bcl-2, cleaved caspase-3 in AC16 cells, and the relative gray scales calculated by ImageJ software. These data were representative results (n = 3) of three repetitions. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. Data are expressed as mean ± SEM.