by Jeffrey M. Perkel
They say death is a part of life. Perhaps nowhere is that more true than at the cellular level, where cell death plays critical roles in development and disease. Human fingers arise from primordial "mittens" via the death of cells that interdigitate the fingers, for instance, while inhibition of cell death in the face of cellular damage can lead to cancer.
"At some point," says Janice Marcelo, cancer product manager at Millipore, "every single customer, whether a neurobiologist or cancer researcher, wonders why cells die."
That death fundamentally occurs in two forms, necrosis and apoptosis. The former is a traumatic form of death in which the cells burst, releasing their contents into the body, inducing an inflammatory response, and possibly damaging nearby cells. The latter, by contrast, is a controlled and tightly regulated process in which the cell effectively dismantles itself piece by piece, until it ultimately can be cleared away by the immune system. In a sense, it's like the difference between destroying a building via a controlled demolition, versus hitting it with a missile. In both cases, the building is gone. But in the former case, collateral damage is kept to a minimum.
Researchers have at their disposal a wide range of tools and techniques for studying apoptosis (and distinguishing it from necrosis), from reagents that detect the morphologic changes inherent in apoptosis, to reagents that monitor the proteins and enzymes that help break down the cell. The former offer a relatively qualitative assessment of cell state – are cells apoptotic or aren't they? – whereas the latter can help tease apart the underlying molecular mechanisms that produced that state. Many are amenable to a range of detection modalities, from microscopy to flow cytometry.
Membrane Assays
One morphologic hallmark of apoptosis is that the cell membrane "flips" – phospholipids normally found on the intracellular face of the plasma membrane bilayer flip to the extracellular face. Researchers can measure this event with reagents specific for these intracellular phospholipids. Annexin V, for example, specifically recognizes phosphatidylserine. When conjugated to a fluorescent dye, the protein provides a simple way to count apoptotic cells, for instance via flow cytometry, as with Roche Applied Science's Annexin-V-FLUOS staining kit. Alternatively, you can use a tagged anti-phosphatidylserine antibody, such as Millipore's Anti-Phosphatidylserine clone 1H6, Alexa Fluor® 488 conjugate.
However, as the membrane's intracellular face is also exposed during necrosis, additional tests are necessary to distinguish apoptosis from necrosis. Larson Manifold, product manager for cellular analysis reagents in the applied science area at Roche Diagnostics, recommends the DNA dye, propidium iodide (PI). "That stains the DNA in necrotic cells, but the DNA in apoptotic cells is not exposed," he says.
As both annexin V (or anti-phosphatidylserine) and PI may be detected via flow cytometry, distinguishing healthy cells from apoptotic cells from necrotic cells is as simple as double staining with both reagents; early apoptotic cells will be Annexin V-positive, PI-negative, whereas necrotic cells are positive for both stains. Some kits, such as Roche's Annexin-V-FLUOS Staining Kit and BD Biosciences' BD Pharmingen™ Annexin V-FITC Apoptosis Detection Kit I, include both reagents.
Sigma-Aldrich's Apo-TRACE™ assays detect another membrane change that occurs during apoptosis – loss of membrane potential, as well as phospholipids scrambling. The Apo-TRACE Apoptotic Cell Staining Kit and Apo-TRACE In Vivo Apoptosis Detection Kit labels apoptotic cells in cell culture and in live animals, respectively, via a fluorescent dye that accumulates in the cytoplasm of apoptotic cells.
DNA Assays
One hallmark of the late stages of apoptosis is fragmentation of DNA into approximately 200 bp pieces. By far the most common approach to measure that event is the TUNEL assay. TUNEL (or "Terminal deoxynucleotidyl transferase dUTP Nick End Labeling") staining uses the enzyme terminal deoxynucleotidyl transferase (TdT) to transfer uridine to the 3' end of the DNA breaks. Because these bases are tagged in some way, for instance using bromodeoxyuridine, they can in turn be detected with a secondary reagent such as a fluorescently labeled antibody. A number of companies offer TUNEL staining kits for both microscopic and flow cytometric analysis, including Millipore's ApopTag® Red In Situ Apoptosis Detection Kit and Guava TUNEL Kit, as well as Life Technologies' Click-iT® TUNEL Alexa Fluor® Imaging Assay.
Millipore also offers an alternative method for detecting DNA breaks. The ApopTag Peroxidase ISOL Apoptosis Detection Kit uses "in situ oligo ligation" (ISOL) to ligate oligos to the blunt ends of DNA characteristic of apoptosis; these oligos are biotinylated, enabling them to capture streptavidin-horseradish peroxidase, which in turn can be detected via a chromogenic substrate.
Roche Applied Science offers an alternative method for detecting DNA fragmentation based, not on 3' end detection, but on cytoplasmic DNA-histone complexes. The Cell Death Detection ELISA PLUS kit, says Manifold, "quantitates the amount of cytoplasmic histone-associated DNA fragments," using a sandwich ELISA in which one antibody captures histones and the other, DNA.
Mitochondrial Assays
Another indication of apoptosis is mitochondrial permeabilization and loss of membrane potential. Millipore's MitoLight® Mitochondrial Apoptosis Detection Kit and flow-based Guava Mitochondrial Depolarization Kit, and BD Biosciences' BD™ MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit use "membrane-permeable lipophilic cationic fluorochromes" to differentially stain healthy and apoptotic cells red and green, respectively, based on mitochondrial membrane integrity. The difference stems from the fact that the dye's fluorescence depends on its aggregation; in the cytoplasm, the dye remains monomeric and therefore green, whereas in mitochondria, the dye aggregates to shift red. Other kits, such as Abcam's Cytochrome c Releasing Apoptosis Assay Kit, measure mitochondrial integrity via release of mitochondrial cytochrome c into the cytoplasm.
Caspase Assays
On an enzymatic level, apoptosis is accompanied by activation of a cascade of proteolytic enzymes called caspases (cysteinyl-aspartic acid proteases). Companies have devised several different approaches to detecting this event.
Roche Applied Science's Fluorimetric Homogeneous Caspases Assay, for instance, uses a fluorimetric substrate to monitor activity of "caspases 2, 3 and 7, [and] caspases 6, 8, 9 and 10 to a lesser extent," according to company literature. The company's Caspase 3 Activity Assay, in contrast, uses an anti-caspase 3-coated microtiter plate to specifically capture that enzyme from a cell lysate. Addition of a fluorogenic substrate produces a fluorescent signal proportional to the level of caspase-3 in the sample.
The use of a capture antibody, says Manifold, is "a unique way of detecting caspase-3 activity."
Promega's luminescence-based Caspase-Glo® 3/7 Assay System uses a pro-luminescent caspase substrate, DEVD-aminoluciferin, to specifically monitor activity of caspases 3 and 7 directly on cultured cells in microwell plates.
According to Pam Guthmiller, Strategic Marketing Manager for cell viability and apoptosis products at Promega, bioluminescent assays have better signal-to-noise than fluorescent assays, and thus better sensitivity and a broader dynamic range.
"Luciferase offers a superior readout over fluorescence," Guthmiller says. "Fluorescent-based assays can be bothered by background fluorescence from media, compounds, or cells."
Millipore's CaspaTag In Situ Apoptosis Detection Kits assess caspase activity in live cells, as opposed to lysates. The kits use fluorescently tagged caspase substrates that enter cells and irreversibly bind to activated caspases.
Antibodies
On a more general level, many companies – among them R&D Systems, BD Biosciences, and Novus Biologicals -- supply antibodies (as opposed to kits) to assay the presence or activity of key apoptotic factors and signaling pathways.
At BD Biosciences, for instance, "we often build reagents that help us better understand the networks or signal transduction pathways important in apoptosis," says Robert Balderas, vice president of biological sciences.
These antibodies may be applied to any of a variety of immunoassays, including western blotting, flow cytometry, fluorescence microscopy, and immunohistochemistry. R&D Systems has compiled 35 antibodies into a microarray; the Human Apoptosis Antibody Array tests for intra-mitochondrial and many other apoptotic proteins simultaneously.
Whatever the assay method used, such antibodies reveal more nuanced information about cell death than do assays like annexin V and TUNEL. Says Hui Li, research and development manager for monoclonal antibodies and apoptosis at Millipore, "Apoptosis is a morphologic change, [but], it is not just one pathway, there are multiple pathways."