What To Look For In An Apoptosis Detection Kit

The mode and timing of a cell’s death, and the pathways it used to get there, can offer insight into the mode of action of pharmaceutical compounds, for example, or allow contributions of particular genes to be teased apart. But it’s important to understand the system you’re working on. Different signals, both internal and external, impinge on a cell—some having more impact on certain cells than on others—and are integrated into the decision whether to undergo apoptosis. As more insult is added, explains Jonathan Morgan of the American Type Culture Collection, more proteins that respond to apoptosis become available, and it becomes more probable that cells are going to die. On the other hand, too much signal, “and the cells don’t have time to go through the nice, orderly process of apoptosis,” he says. “They actually just necrose; explode.”

Similarly, the timing of measurements is important. Some cells may undergo apoptosis relatively soon after an insult, “and if you don’t time your experiments correctly you’ll miss it,” says Morgan.

Factors such as the density at which cells are plated, and even the density of the stock from which they were drawn, can affect the sensitivity to, and the timing of, apoptosis, “illustrat[ing] the importance of understanding the kinetics of cell death in the model system being used before choosing a time to collect data,” wrote Terry Riss and Richard Moravec of Promega.

Researchers utilize many different methods to measure whether apoptosis has occurred, and at which point in several processes apoptosis was initiated. Some make use of sophisticated instrumentation; others use off-the-shelf kits and standard laboratory equipment. Which method your lab chooses should depend on what you are investigating, how many answers you need and how fast you need them, the equipment your lab has, and the time and money it has to invest. Here are a few considerations to help make the decision easier.

The various intracellular pathways that can lead to apoptosis offer a number of ways to measure the phenomenon, as well as to determine the specific mechanism involved and how far into the process a cell has gone: membrane blebbing and apoptotic bodies can be observed microscopically; the characteristic 180 base pair “DNA ladder” can be seen on agarose gels, or assayed for by TUNEL staining (which labels the nicked ends of DNA); there are kits to detect activation of various caspase enzymes, Cytochrome C release, and PARP cleavage; membrane asymmetry can be measured by staining for annexin 5; protocols exist for flow cytometry and laser scanning cytometry, for immunocytochemistry and immunohistochemistry, for western blotting, and for ELISA; frozen and preserved tissue, cell cultures, and primary isolates can be used; and so on.

William Couldwell, whose University of Utah lab studies apoptosis in neural cancers, always looks at two different mechanisms to assure himself that what he’s seeing is truly apoptosis. Reviewers, too, like to see confirmation.

But “keep it simple unless you’re trying to look for mechanisms,” advises Couldwell, “the simple techniques should be up and running, and core for any molecular biology lab these days.”

For more involved measurements, Couldwell’s lab uses a $500,000 laser scanning cytometer (LSC)—which can automatically query several parameters of individual cells on a microscope slide simultaneously (including morphology), at the same time integrating the results from large numbers of cells. He calls LSC the “Rolls Royce” for this kind of work, and acknowledges that it’s not practical for most labs. But, he says, “as a core facility in a big institution, that would be very smart.”

There are also other, more specialized cell imagers. Cellomics, for example, offers a high-throughput hardware and software package for acquisition and quantitative analysis of cells in microplates. Their multi-parameter apoptosis kit—which can be used with their automated system or without it—can determine whether a cell is just beginning to undergo apoptosis, or in a more intermediate or terminal state, says Sarah Tencza, product manager for Bioapplications. “So you can say, based on that pattern of staining across the three fluorescent dyes, what stage of apoptosis each cell is in.”