The caspase family of cysteine proteases play a key role in apoptosis and caspase-3 is the most extensively studied apoptotic protein among the family members. Caspase 3 is synthesized as an inactive pro enzyme that is cleaved into an active form in cells undergoing apoptosis. Quantification of cleaved caspase-3 is useful for further investigation of a toxic effect induced by a test substance. The objective of this assay is to evaluate the test article for their potential to induce apoptosis in vitro in mammalian cells. The assay can be performed in accordance with Good Laboratory Practices.
For the Cleaved Caspase 3-Test, L929 cells are seeded into 96-well plates and maintained in culture for 24 h (~ 1 doubling period) to form a semi-confluent monolayer. Cells are then exposed eight test compound concentrations covering a large range. Cisplatin is used as a positive control. After 24 h exposure, proteins are extracted from cells and incubated with the substract DEVD-pNA. This substrate is cleaved by active caspase 3. The pNA light emission is measured by spectrometry at 400 nm. The absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in Caspase-3 activity.
Experiment will start after receipt of signed protocol and the test substance. A draft report will be ready for review within 4-6 weeks after the experiment start date. Documents will be archived for six years.