The Promega Caspase-Glo® 3/7 Assay System is a homogeneous luminescent method used to measure caspase-3 and -7 activities. Caspase 3 and 7 are members of the cysteine protease family and are essential components of apoptotic pathways in mammalian cells. The Caspase-Glo 3/7 Assay System contains a single reagent solution which includes a luminogenic substrate for caspases 3 and 7, a thermostable luciferase, and buffer to induce cell lysis. The presence of caspase 3 or 7 cleaves the luminogenic substrate which in turn leads to the release of the substrate for luciferase resulting in the production of light.
The Caspase-Glo® 3/7 Assay is ideal for use with multiwell-plate formats as the procedure involves the addition of a single reagent directly to cells cultured in their regular serum-containing media. No further steps are required, such as cell washing or removal of media. Addition of the single reagent lyses the cells and results in the generation of a luminescent signal which is proportional to the amount of caspase activity present. The caspase and luciferase enzyme activities reach steady state in approximately one hour and this activity is maintained for several hours with a minimal loss of signal. The sensitivity of the assays is as low as 20 apoptotic cells. Promega report that it is possible to multiplex this assay with other homogeneous assays to measure more than one parameter from a single well.
We have used this assay primarily to measure apoptosis in cancer cell lines (HeLa, Colo205, SW620, HCT116 etc.) treated with cytotoxic agents (e.g. Taxol) in a 96-well format. The protocol for the assay involves initially plating cells at the desired cell concentration (100 µl volume per well) in opaque-walled multiwell plates, suitable for use in a luminometer, in their regular culture medium. Cells are treated in accordance with the experimental protocol (e.g. addition of various concentrations of cytotoxic agents and controls are included, such as cells which receive no treatment and wells containing medium without cells in order to obtain a value for background luminescence. This control is important as there is often caspase activity in the serum present in the media). The cells are then incubated according to experimental protocol. Once the assay incubation is complete, the plates are removed from the incubator and allowed to equilibrate to room temperature for approximately 30 minutes. During this incubation at room temperature, the Caspase-Glo® 3/7 Reagent Buffer is thawed out and added to the lyophilized Caspase-Glo® 3/7 Reagent. Once the plate has equilibrated to room temperature, an equal volume of Caspase-Glo® 3/7 Reagent is added. The plate contents are then mixed on an orbital shaker for 2 minutes to lyse the cells. The plate is allowed to incubate at room temperature for a further 1-3 hr to allow the luminescent signal to develop. At this point, the luminescence is recorded.
We have found this assay to be very consistent and easy to use; however, a number of variables may require optimization for each cell line. Most importantly the cell number needs to be optimized as caspase activity varies quite considerably from one cell line to another. When we employ a new cell line, we have always checked to ensure that the Caspase-Glo® 3/7 Reagent lyses the cell line by lysing cells in optically clear plates and checking microscopically prior to moving to opaque plates. In addition, it is necessary to optimize the incubation time to allow the luminescent signal to develop.
In conclusion, this is a simple assay to measure apoptosis in cell lines. The homogeneous format plus ease of optimization are a great advantage.
Senior Research Scientist
Discovery Biology
Vitae Pharmaceuticals