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 modifications17 (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.
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.
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