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Fluorogenic Caspase-3 activity assay

YH Yi-Ta Hsieh
HT Hsi-Feng Tu
MY Muh-Hwa Yang
YC Yi-Fen Chen
XL Xiang-Yun Lan
CH Chien-Ling Huang
HC Hsin-Ming Chen
WL Wan-Chun Li
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Cells were seeded in 96-well plate for 24 h, rinse cell with ice-cold PBS and spin the plate at 300xg for 10 min. Cell lysates were collected by cell lysis buffer on ice for further use. Caspase-3 activity was detected following the manufacturer’s instructions (Cell signalling, Kit#5723). N-Acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) is used as the fluorogenic substrate while activated Caspase-3 cleaves this substrate between DEVD and AMC, generating highly fluorescent AMC that is detected using TECAN SPARK multimode microplate readers at IRC, NYCU. Fluorescence intensity was measured with excitation wavelength at 380 nm and emission wavelength at 450 nm at 37 °C.

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