Caspase-3 colorimetric assay

摘要

The commonly used ApoAlert caspase-3 colorimetric assay kit detects a cleaved chromophore p-nitroanilide (pNA), after cleavage from a labeled substrate DEVD-pNA (1-6). The kit was used for murine bone marrow-derived mast cells (3) and human Burkitt lymphoma (Daudi) cells (5) with apoptosis induced by cytokine depletion and antisense p21 transfection, respectively. In comparison to the uninduced level, a 3.3-fold (3) or 5-fold (5) increase of caspase-3 activity was observed in the induced samples. According to the product analysis certificate, a minimum of 3-fold increase in caspase-3 activity was detected after induction of Jurkat cells in which apoptosis was induced by 40 #mu#M actinomycin D for 15 h in comparison to uninduced controls. In our study with breast cancer cells and arsenite-induced apoptosis, we obtained a less than 3-fold increase. Suspecting that an unknown reaction product interfered with the spectrophotometric detection of the pNA at 405 nm, we developed a modified procedure that increases the sensitivity of the kit for our arsenite-induced breast cancer cells. The procedure was modified to the following: after incubation of the reaction at 37 deg C for 1 h, the reactions were boiled at 100 deg C for 5 min and then centrifuged at 20000x g for 10 min to precipitate debris. Caspase activity was determined spectrophotometrically at 405 nm by measurement of the free pNA produced by cleavage of the substrate in the supernatant. Quantitation of pNA was determined by comparison to a pNA standard curve. Data are expressed as the X + SEM derived from three separate experiments.