Caspase 3 assay kit

The activity of caspase-3 activity was detected using Caspase-3 assay kit (Beyotime Institute of Biotechnology) according to the protocol of manufacturer. Briefly, transfected cells after 4 Gy IR were collected and added into 10 μl of reaction buffer with the kit, followed by incubation for 2 h at 37 °C. Then the optical density value was measured at the wavelength of 400 nm. The relative caspase-3 activity of each sample was calculated as the absorbance of the well by that of the control group.

The Caspase-3 activity in transfected C666-1 cells was evaluated using Caspase-3 Assay Kit (Beyotime, China) referring to the manufacturer’s instruction. In brief, transfected cells were dissolved with buffer solution, and then lysate was collected and centrifuged for 10 min at 1,500 × g. Then 100 mL of Caspase-3 reagent was added to the supernatant and incubated at 37°C for 2 h. After that, optical density (OD) at 405 nm was measured using microplate reader (BioTek, VT) to determine Caspase-3 activity.

Caspase 3 activities were performed using caspase 3 Assay Kit (Beyotime) according to the manufacturer′s instructions. H9‐CMs were digested with Trypsin‐EDTA (Gibco) and collected by centrifugation at 600 g for 5 minutes at 4°C. The cell pellets were washed with DPBS (Gibco) and re‐suspended in 1 × lysis buffer at a concentration of 100 μL per 2 million cells, incubated on ice for 15 minutes and then centrifuged at 16 000‐20 000 g for 10‐15 minutes at 4°C. Appropriate amount of protein was put in a 96‐well plate, and 10 μL of Ac‐DEVD‐pNA (acetyl‐Asp‐Glu‐Val‐Asp p‐nitroanilide) (2 mmol/L) was added per well and then incubated for 60‐120 minutes at 37°C. Absorbance at 405 nm was read using a MD M5 SpectraMax reader (Molecular Devices).

Caspase-3 activity levels in isolated cerebral microvessels as well as murine cerebrovascular endothelial cells were assayed as previously described by Yin et al. with minor modifications^{17 (link)}. Briefly, 1 × lysis buffer was applied to lyse cerebrovascular endothelial cells. Following 10 min on ice, the cell lysate was spun down for 10 min at 10000 g (4 °C). The resulting supernatant was subjected to a caspase-3 activity assay according to the kit’s instructions (Caspase-3 Assay Kit, Beyotime Institute of Biotechnology). A microtiter plate reader was used to read optical density (OD) values at 405 nm. Relative caspase-3 activity was calculated by comparing OD levels from the experimental groups against those of controls.
DNA fragmentation levels in isolated cerebral microvessels were assayed as previously described by Yin et al.^{17 (link)}. Briefly, we first extracted the genomic DNA from isolated cerebral microvessels. Then, a commercial apoptotic DNA-ladder kit (Roche) was employed to measure DNA fragmentation levels.

Alizarin Red S was performed to visualize the formation of mineralized matrix (Shanghai Gefan Biological Technology Co., Ltd, Shanghai, China). The calcium content was determined using a calcium colorimetric assay kit (Nanjing Jiancheng Biological Engineering Institute, Nanjing, China). Alkaline phosphatase (ALP) activity was measured using an ALP kit (Beyotime Institute of Biotechnology, Shanghai, China). Caspase-3 activity was analyzed using a caspase-3 assay kit (Beyotime Institute of Biotechnology, Shanghai, China).