The term “apoptosis” refers to a series of cellular events that culminate in the intentional death of a cell. While apoptosis plays an important role in many non-pathological functions such as growth, differentiation and development of the immune system, it can also be a major player in pathological phenomena such as cancer, where specific elements of various apoptotic pathways may be defective. In these instances, cells that would normally be slated for death continue to grow and divide. Hence, the reactivation of apoptosis is being actively examined as a potential therapeutic strategy for cancer.
Some of the key players in apoptosis are members of a family of cysteine aspartic acid-specific proteases called caspases. Caspases participate in a proteolytic cascade that results in the ordered destruction of the cellular survival system. Some caspases function early in the cascade and are referred to as initiator caspases (e.g. caspases 8 and 10) and others function much later and are called effector caspases (e.g. caspases 3, 6, and 7). So, despite the complexity of apoptosis, it is possible to gauge the level of ongoing apoptosis in a population of cells by measuring the activity of the effector caspases. Promega (Madison, WI) now offers a kit that permits the activity of two effector caspases, caspases 3 and 7, to be measured using a very simplified assay.
Promega’s assay, the Apo-ONE Homogeneous Caspase-3/7 Assay, works by providing cells with a substrate that fluoresces following cleavage by the activated caspases 3 and 7. What makes the assay so simple is that, before use, the profluorescent substrate gets diluted into a buffer that not only permeabilizes cells in culture, but also supports optimal caspase 3/7 activity. The basic assay protocol is to culture cells with or without the desired apoptosis inducer (e.g. serum withdrawal or radiation-induced DNA damage), add the diluted substrate, incubate and read using a fluorescent plate reader. For cell lines that do not lyse easily, you can include a freeze/thaw step without affecting the results. The assay can also be used on purified caspase preparations.
I recently used the Apo-ONE Homogeneous Caspase-3/7 Assay to measure apoptosis initiated by serum withdrawal on two cell lines. One cell line stably expressed an siRNA against a protein that we have been studying and the other expressed the scrambled siRNA sequence as negative control. 
Both cell lines are normally cultured in RPMI-1640/10% FBS, but for the assay I switched them to RPMI-1640/0.5% FBS for 4 days. On day 4, I harvested the cells, counted them and transferred serial dilutions to a black 96-well tray. I then added an equal volume of the diluted substrate, put the plate at –80°C for a few minutes, thawed, mixed and incubated the plate at RT for 2 h. I then read the plate at an excitation wavelength of 485 nm and emission at 590 nm. As shown in the figure, the knockdown cells show elevated caspase 3/7 activity as compared to the scrambled control cells. This agrees with our results in nude mouse xenografts of the two cell lines using a TUNEL assay.
In summary, Promega’s Apo-ONE Homogeneous Caspase-3/7 Assay offers a simple and sensitive method to gauge apoptosis by caspase 3/7 activation. Although it is a bit pricey for 96-well assays (about $3/well), it is also amenable to 384-well format which brings it to less than a dollar/well.
Michael Campa, Ph.D.
Assoc. Research Professor of Radiology
Duke University Medical Center