Measurement of Caspase-9 and Caspase-3 Activity

As described previously by our laboratory, caspase-9 and caspase-3 activity was measured by incubation of the proteins fluorometric substrates Ac-DEVD-AMC and Ac-LEHD-AMC, respectively (Lu et al., 2016). Briefly, the reaction was initiated by mixing equal amount of the proteins (30 μg) from cortex (S1) and hippocampal CA1 with specific substrate in the assay buffer and incubated for 1 h at 37°C. The fluorescence of free AMC, corresponding to the proteolytic activity of caspase, was measured on a spectrophotometer (Perkin Elmer). Relative caspase activity in each sample was expressed as the changes of fluorescent units and compared between groups.