Cell proliferation was assessed with Cell Counting Kit-8 (CCK-8) (Labgic Technology Co., Ltd., Beijing, China) reagent following the manufacturer’s instructions. CFs were passaged in 96-well plates at the density of 8 × 103 cells/well. After culturing for 24 h, cells were treated with serum-free medium for another 24 h and then were placed on three-gas incubators environment (96% nitrogen, 5% carbon dioxide and 1% oxygen). After treatment with hypoxia for 12 h, 20 ul of CCK-8 reagent was added into each well and CFs were incubated at 37°C for another 2 h. The optical density was measured at a wavelength of 450 nm. Serum-free mediums in normal incubators served as the negative control.
The apoptosis of NRVMs after exposed to hypoxia was assessed by staining cells with Annexin V-fluorescein isothiocyanate (APC) Apoptosis Detection Kit (BD Biosciences). The cells were collected and then washed with cold PBS twice. Thereafter, they were resuspended in 100 ul of Annexin V binding buffer and incubated with 5 ul of APC-conjugated Annexin V and 5 ul of propidium iodide for 15 min in the dark. Annexin V binding buffer (200 ul) was then added to each tube. Finally, the cells were examined using a BD FACS-Canto II flow cytometer (BD Biosciences, CA). All experiments were repeated three times, independently.
The apoptosis of NRVMs after exposed to hypoxia was assessed by staining cells with Annexin V-fluorescein isothiocyanate (APC) Apoptosis Detection Kit (BD Biosciences). The cells were collected and then washed with cold PBS twice. Thereafter, they were resuspended in 100 ul of Annexin V binding buffer and incubated with 5 ul of APC-conjugated Annexin V and 5 ul of propidium iodide for 15 min in the dark. Annexin V binding buffer (200 ul) was then added to each tube. Finally, the cells were examined using a BD FACS-Canto II flow cytometer (BD Biosciences, CA). All experiments were repeated three times, independently.