A luminescent assay which measures caspase-3 and -7 activities is employed to detect these important members of the cysteine aspartic acid-specific protease (caspase) family.
Caspases play key effector roles in apoptosis in mammalian cells, activating endonucleases and cleaving substrates such as PARP, which then leads to the morphological changes associated with apoptosis. The assay uses a luminogenic caspase-3/7 substrate to detect caspase activation, in a reagent optimised for caspase and luciferase activity as well as cell lysis. Addition of a single caspase 3/7 reagent in an “add-mix-measure” format results in cell lysis, followed by caspase cleavage of the substrate and generation of a “glow-type” luminescent signal, produced by luciferase. Luminescence is proportional to the amount of caspase activity present.
Cells are exposed to test chemical for the required number of time points. Typically this will include time points at 16 and 24 hours, with additional time points included as required. Samples are removed from the cell cultures and mixed with an equal volume of the caspase reagent and incubated for 90 minutes at room temperature.
After the incubation, luminescence is assessed using a luminometer. Data can be presented as total luminescence values or can be normalised to cell densities if required.