Ac lehd amc

Caspase-3 and caspase-9 activities in the cytosolic protein fractions were measured using fluorometric substrates as previously described [46 (link)]. The Ac-DEVD-AMC and Ac-LEHD-AMC (AnaSpec) are substrates for caspase-3 and caspase-9, respectively. The fluorescence of free AMC was determined with an excitation wavelength of 360 nm and an emission wavelength of 460 nm, in a Synergy HT Microplate reader (BioTek Instruments). The values were expressed as fold change in fluorescent units compared with control group.

Hippocampal CA1 regions were quickly separated on an ice pad along the hippocampal fissure, using a standardized microdissection procedure. The separated tissues were homogenized by using a motor-driven Teflon homogenizer in ice-cold homogenization buffer with inhibitors of proteases and enzymes as previously described [21 (link)]. Caspase activities for caspase-3, -8, and -9 were measured in the protein homogenate using fluorometric substrates. The following substrates were used for caspase-3, -8, and -9, respectively: Ac-DEVD-AMC, Ac-IETD-AMC, and Ac-LEHD-AMC (AnaSpec, Fremont, CA). The substrates were cleaved proteolytically by the corresponding caspases, and the fluorescence of free AMC was measured. For determination of caspase activities, 100 μg of total protein were incubated for 3 h in caspase buffer (100 mM HEPES, 10% sucrose, 10 mM DTT, 0.1% CHAPS, 1 μg/ml leupeptin, and 1 mM PMSF) with 100 mM substrate. Fluorescence was determined with an excitation wavelength of 380 nm and an emission wavelength of 460 nm for AMC using a Synergy HT Microplate reader (BioTek Instruments Inc, Winooski, VT). Values are expressed as fluorescence of AMC per μg of protein and then were compared with sham group.

Caspase-3 and caspase-9 activities were measured in cytosolic protein fractions using fluorometric substrates as previously described by our laboratory (Lu et al., 2016 (link)). The substrates for caspase-3 and caspase-9 were Ac-DEVD-AMC and Ac-LEHD-AMC (AnaSpec, Fremont, CA, USA), respectively. The substrates were cleaved proteolytically by the corresponding caspases, and the fluorescence of free AMC was measured. Fluorescence was determined with an excitation wavelength of 360 nm and an emission wavelength of 460 nm for AMC, in a Synergy HT Microplate reader (BioTek Instruments Inc, Winooski, VT, USA). Values were expressed as changes in fluorescent units per microgram of protein and presented as percentage of control group.