by Caitlin Smith
Eukaryotic cells have a dramatic way of removing themselves when their time has come. A pre-planned cellular suicide known as programmed cell death, or apoptosis, is spelled out by detailed pathways that we are still uncovering. Apoptotic cells undergo changes in morphology and biochemistry—such as membrane blebbing, or DNA fragmentation. Apoptosis assays utilize these and other characteristics. Apoptosis assays are wide-ranging, partly because the process itself can be divided into early, middle and late stages depending on which pathways are studied. Here we focus on five main types of apoptosis assay looking at: caspase, DNA fragmentation including TUNEL and DNA ladder analysis, annexin V and cell permeability, mitochondria, and apoptosis signaling pathway analysis.
Why Use Caspase Assays?
Caspases are a group of aspartic acid-specific cysteine proteases that are active in the early stages of apoptosis, when they initiate a cascade of biochemical events leading to the disabling of multiple cell functions. Caspases can be measured by in vitro enzyme assays or by cleavage of an in vivo caspase substrate. In general, mammalian caspases can be grouped by function: cytokine activation includes caspases 1, 4, 5, 13; apoptosis initiation includes caspases 2, 8, 9, 10; and apoptosis execution utilizes caspases 3, 6, 7. Caspase assays work well when you need a sensitive quantitative assay to detect apoptosis in its very early stages.
“One of our main focuses is on the caspases, since they range throughout the apoptotic pathway, and [the individual caspases] are unique to different parts of the pathway,” says Lauren Buck, associate marketing manager at Roche. “We offer a homogeneous caspase assay, which is broad enough to encompass all caspases in one assay, to detect a general apoptotic event. Then researchers can delve down specifically using kits with more specific markers.”
Buck says that traditional studies of apoptosis are hindered by the fact that they take measurements at a single point in time. “Roche’s xCELLigence System opens the door to a more holistic view of the apoptotic pathway, allowing more insight into what’s actually going on inside the cells.” The ability of the xCELLigence System to make real-time measurements enables researchers to pinpoint the best time to perform endpoint assays. The xCELLigence System uses label-free impedance technology, says Buck, “in which a very low voltage is passed through cells on a 96-well plate with gold micro-electrodes, and the instrument is able to provide kinetic profiles based on impedance measurements, and from there characterize growth curves and morphology changes.”
If you want to use a caspase assay to measure activity in live cells, OncoImmunin offers cell-permeable fluorogenic substrates for detection by flow cytometry, microscopy, and other high-throughput techniques. “The uniqueness of these assays lies in their capability [to measure] the actual protease activity of individual caspases in their physiological environments, compared with other probes which are either suicide inhibitors or are cell-impermeable antibodies. Neither allows measurement of protease activities,” [I don’t believe in changing the content of quotes, but I felt compelled to change the punctuation in this one, and particularly to delete the parentheses.]says Beverly Packard, president of OncoImmunin. “Our probes' uniqueness is possible due to the following properties: One, probes are highly cell permeable with an achieved intracellular concentration of up to 2 uM; two, high cell permeability is uniform for all cells and tissues; three, judicious selection of a range of fluorophores allows multiplexing; four, lack of probe cytotoxicity enables continuous observation over several days; and five, live-cell assays based on this technology have been shown to be suitable for high-content and high-throughput drug candidate screening.” [I’m assuming this is the end of the quote … ?]
OriGene also offers its Prizm™ Fluorogenic Cell Assay Kits for live apoptosis assays, using cell-permeable fluorogenic substrates for monitoring enzymatic activity in biological samples. “The main difference [in our] caspase assay kits is that our Prizm assays allow protease activity monitoring in living cells under a fluorescence microscope, allowing real-time and dynamic activity monitoring,” says Walter Tian, vice president of marketing at OriGene.
Why Use DNA Fragmentation Assays?
A hallmark of apoptosis is DNA cleavage, so measuring the resulting fragments as DNA ladders on a gel, with the rungs comprised of 180 base pair (bp) multiples, can be a useful and straightforward method of determining apoptosis in cell populations. DNA fragmentation can also be measured using ELISA quantification of histone-complexed DNA fragments.
Besides DNA ladder analysis and ELISA, TUNEL assays also are used for measuring DNA fragmentation. TUNEL (terminal deoxynucleotidyl transferees (TdT)-mediated dUTP nick end labeling) detection kits work for several sample types, including adherent cells, sections of paraffin-embedded tissue and cryopreserved tissue sections. Advantages of the TUNEL assay are its sensitivity and that it gives a large range of quantitative measurements over several orders of magnitude. Disadvantages include possible radiolabeling and a lot of washing of target cells.
Promega’s DeadEnd™ Systems offer a kit for quantifying apoptotic cells within a population mixed with non-apoptotic cells. The system end-labels fragmented DNA using a nonradioactive TUNEL assay. GenScript also offers its TUNEL Detection Kits, both colorimetric and fluorescent. Life Technologies additionally sells kits for measuring chromatin condensation and DNA fragmentation. Likewise, Roche offers related kits, such as its Apoptotic DNA Ladder Kit, Cellular DNA Fragmentation ELISA, Cell Death Detection ELISA, and In Situ Cell Death Detection Kit.
Why Use Annexin V
and Cell Permeability Assays?
Annexin V is another popular marker for middle-stage apoptosis, because within the cytoplasm it binds to phosphatidylserine, which translocates to the outer surface of the plasma membrane during apoptosis. Thus phosphatidylserine can be detected through staining of annexin V, a membrane protein forming voltage-dependent calcium channels.
BioVision offers a range of apoptosis tools that allow detection at different stages and even in different compartments of a cell. The company’s products also “allow researchers to determine apoptosis activation pathways, such as mitochondrial-mediated, ligand-receptor-mediated or stress-mediated pathways,” says Gloria Zhang, product manager at BioVision. She notes that the type of assay to use depends on the research and sample. “If live cells are being used, then flow cytometry- or microscopy-based assays [such as annexin V assays and CaspGlow assays] are good choices,” says Zhang. “If cell lysates are being used, BioVision’s fluorometric or colorimetric caspase assay kits are desirable. If tissue slides are being used, then BioVision’s ApoBrdU-IHC kit is one of the best choices.”
Why Use Mitochondrial Assays?
Mitochondrial assays for early-stage apoptosis use a cationic dye that typically fluoresces one color in healthy cells and another color in apoptotic cells. In healthy cells, for example, the dye may accumulate in the mitochondria, where the dye fluoresces red. In apoptotic cells, by contrast, the mitochondrial membrane has a different membrane potential, which makes the dye stay in the cytoplasm, where it glows green. This assay would be suitable for fluorescence microscopy or for flow cytometry using two channels for the two colors.
Life Technologies’ Apoptosis Assay Kits are designed to detect multiple apoptotic parameters in a single flow cytometry assay, to dissect the early and middle stages of the apoptotic response. “Life Technologies’ MitoTracker dyes are membrane potential-dependent probes for staining mitochondria in living cells which can be fixed,” says Jolene Bradford, R&D associate director for flow cytometry systems at Life Technologies. “And the MitoProbe dyes provide quick and easy detection of the loss of mitochondrial membrane potential.” Life Technologies offers a range of assays for detecting apoptosis at different stages using flow cytometry and recommends using more than one test. “No single parameter fully defines cell death in all systems,” says Bradford. “Therefore, it is often advantageous to use a multi-parametric approach when studying apoptotic events and their temporal relationships. Cells can be selectively targeted with fluorescence probes to characterize the progression toward cell death.”
Sallie Cassel, director of product marketing at EMD Millipore, says, “We are seeing a strong upsurge in the study of mitochondria and their impact on apoptosis. EMD Millipore has recently launched a number of new mitochondrial-related products from antibodies to isolation kits and mitochondrial flow cytometry kits.” EMD Millipore also offers apoptosis products “across a range of detection platforms: antibodies for Western blotting, immunohistochemistry and immunocytochemistry; the ApopTag line of TUNEL assays to detect DNA fragmentation and a range of caspase activity assays; Milliplex multiplex assays for Luminex xMAP systems; and FlowCellect kits optimized for our guava benchtop flow cytometers.”
New Tools to Consider
SABiosciences, a Qiagen Company , emphasizes the importance of studying different factors in a given pathway, and their relationships with one another. The RT2 Profiler PCR Arrays facilitate the expression analysis of a pathway-focused set of genes. “By pathway-focused, I mean that we have examined certain pathways, either of biological events, classes of genes or those related to diseases, to identify the most relevant pathway-specific genes and allow for their easy analysis with our PCR Arrays,” says Shankar Sellappan, Ph.D., global product manager at SABiosciences, a Qiagen Company. The company offers apoptosis and related arrays for pathway-focused apoptosis study. “As we strongly believe in performing pathway-focused research, we believe scientists should consider how their samples may be affected by the treatments/conditions that may lead to apoptosis,” notes Sellappan. “Are they looking at upstream or downstream markers of apoptosis? What is their aim in analyzing the expression of the signaling participants? Which stage of apoptosis is important to them, and if the stage is important, are they collecting their samples at the correct time and stabilizing the samples so as to maintain the molecular signals for later analysis?”
Sellappan says that for someone just starting out in the arena of apoptosis, who may not have specific targets yet, “we would recommend a screening assay, for example, the Apoptosis Pathway RT2 Profiler PCR Array,” he advises. “This might provide some immediate clues as to which genes are potentially up-regulated in their samples that may lead to apoptosis. Then the researcher can select just those genes that are modulated and study them with our Custom RT2 Profiler PCR Arrays on a greater number of samples, use our Methyl-Profiler DNA Methylation PCR Arrays to monitor epigenetic changes that may influence apoptotic events or our CIGNAL Reporter assays to monitor apoptosis pathway activities.”
Tian, of OriGene, agrees that a single time point view of apoptosis is not the best one. “In addition to regular needs for assay sensitivity, specificity, overall assay time and cost, one suggestion is to study apoptosis as a biologic process,” he says. The “traditional approach of measuring apoptosis at an endpoint is no longer sufficient. Ideally one should pick an assay that allows dynamic monitoring, studying apoptosis as it relates to inducing stimuli and over time.”
The image at the top of this article is Roche Applied Science's xCELLigence System.