The techniques used for measuring and observing apoptosis

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The number of cells within an organism is tightly regulated- not simply by controlling the rate of cell division, but also by controlling the rate of cell death. If cells are no longer needed, they commit suicide by activating an intracellular death program. (Liao, 2008)

Apoptosis, is defined by distinct morphological and biochemical changes mediated by a family of cysteine aspartic acid-specific proteases (caspases), which are expressed as inactive precursors or zymogens (pro-caspases) and are proteolytically processed to an active state following an apoptotic stimulus. (Liao, 2008)

The aim of this paper is to discuss the techniques for measuring and observing apoptosis, whilst commenting on any limitations reported to date.


The intracellular machinery responsible for apoptosis

Kerr, Wylie and Currie (1972) observed controlled cell death that was distinct from uncontrolled necrotic death. They noticed a characteristic, identical sequence of events in many different types of cells and published their observations in 1972 and used the term apoptosis. (Alberts, 2007; Cancer, 1972)

In apoptosis, cell shrinkage and membrane ruffling (blebbing) occur, and the cell disintegrates into small membrane-bound apoptopic bodies. Inside the cell chromatin condensation and nuclear fragmentation occur, which are accompanied by breakdown of the DNA into regular size fragments. On the surface of the cell lipids are rearranged in the bilayer of the plasma membrane with the lipid phosphatidylserine becoming exposed to the outside. (Hancock, 2005)

Measuring and observing apoptosis

Apoptosis is essentially a morphological state arrived at by a whole variety of different biochemical pathways. Some routes may result in the expression or loss of an antigen but there is no assurance that the same biochemical alterations occur in every cell. The identification of protein markers permits ready detection by conventional immunohistochemistry, which facilitates rapid and confident assessment of apoptosis. (Harrison, 1996)

The in situ end labelling and in situ nick translation techniques rely on the presence of DNA strand breaks characteristic of the nuclear implosion and fragmentation seen in apoptosis. Thus, labelling with biotinylated nucleotides and subsequent immunodetection can be used to identity sensitively cells with strand breakage. (Harrison, 1996)

Activation of the caspase-3 pathway is a hallmark of apoptosis and can be used in cellular assays to quantify activators and inhibitors of the death cascade by molecular identification. The response is both time and concentration dependent suggesting that multiple pathways play a role in triggering the caspase-3 activation. (Bio Medicine, 2008)

TUNEL assay is a technique used for observing activation of the caspase-3 pathway by biochemical verification. The enzyme TdT is able to add nucleotides to the ends of DNA fragments; most commonly, biotin-labelled nucleotides (usually dUTP) are added. The biotinylated DNA can be detected by using streptavidin, which binds to biotin, coupled to enzymes that convert a colourless substrate into a coloured insoluble product. Cells stained in this way can be detected by light microscopy. (Janeway, 2001)

The cells with unsuccessful DNA repair may undergo apoptosis; in some cases, false positive labelling may result. TUNEL staining has been reported to give false positive staining in the kidneys of nude and BALB/c mice. In situ localization of activated or cleaved caspase-3 is starting to replace TUNEL as the molecular verification of the apoptotic process. (Hughes, 2007)

Extrinsic and Intrinsic signal activation

The extrinsic pathway is initiated by ligation of transmembrane death receptors (DR) with their respective to activate membrane-proximal caspases, which in turn cleave and activate effector caspases. A killer lymphocyte carrying the Fas ligand binds and activates Fas proteins on the surface of the target cell. Adaptor proteins bind to the intracellular region of aggregated Fas proteins, causing the aggregation of procaspase-8 molecules. These then cleave one another to initiate the caspase cascade. This pathway can be regulated by c-FLIP, which inhibits upstream initiator caspases, and inhibitor of apoptosis proteins (IAPs), which affect both initiator and effector caspases. (Alberts, 2007; Liao, 2008)

The intrinsic pathway requires disruption of the mitochondrial membrane and the release of mitochondrial proteins, such as cytochrome c. Cytochrome c, which binds to and causes the aggregation of the adaptor protein Apaf-1. Apaf-1 binds and aggregates procaspase-9 molecules, which leads to the cleavage of these molecules and the triggering of a caspase cascade. (Alberts, 2007, Liao, 2008)

The primary regulatory step for mitochondrial-mediated caspase activation might be at the level of cytochrome c release. The known regulators of cytochrome c release are Bcl-2 family proteins. Members of this family are divided into two main groups, the anti-apoptotic proteins with Bcl-2 and Bcl-Xl as archetypes and the pro-apoptotic proteins such as Bax. The ratio between these different proteins determines the sensitivity of the cell to apoptosis; antiapoptotic proteins inhibit apoptosis by counteracting Bax and Bak, and BH3-only proteins either trigger apoptosis through direct interaction with Bax or sensitize cells to death by inactivating Bcl-2 or Bcl-Xl. (Cartron, 2003; Liao, 2008)

RT-PCR primer sets that are specific for genes involved in inducing and regulating the apoptotic response. These primers are specific for genes encoding proteins from the Fas and Fas ligand, Bcl-2 and ICE protein families. The RT-PCR primer sets for studying the apoptotic response are designed to meet several criteria. The primers sets, based on known genomic sequences, amplify a region that spans at least one intron. To distinguish the amplification products from genomic sequences, which are longer than the cDNA products, the primer sets amplify PCR products that are 400 bp to 650 bp in length. The primers are synthesized as 18- to 27-mer oligonucleotides. Each set of primers amplifies only a specific target. (Biomedicine, 2008)

Direct measurement using fluorescence resonance energy transfer has shown the interaction between Bax and Bcl-2. (Cartron, 2003)

Light microscopy and electron microscopy, including staining, are frequently used to observe the morphological changes of the cells undergoing apoptosis. There are many staining protocols used for identification of apoptotic cells, and the choice varies subject to the laboratory and the tissue being studied. (Wang, 2008)

The advantages of Near-Field Scanning Optical Microscope (NSOM) are observing in normal environment, observing in nanometre scale resolution, and observing in non-contact mode. (Wang, 2008)


Using morphology, biochemical or molecular methods to identify, localize and quantify apoptosis gives strength to many research studies. The measurement of the level of apoptosis within tissue sections represents only a ‘snapshot’ of one time point during a developmental, physiological or pathological process. Given the rapid nature of apoptosis and its cryptic nature in tissue sections, these measurements may often be underestimates of the actual extent of apoptosis. The ability to measure the levels of apoptosis within living organisms, including humans, non-invasively at repeated intervals over time would facilitate the analysis of apoptosis in many organs as well as tumours. (Hughes, 2007)

These investigative techniques have broadened the understanding of disease development and will undoubtedly present real opportunities for novel therapeutic intervention. For example, the gene-driven nature of apoptosis and its modulation by various controlling molecules have provided a basis to develop therapies for selectively protecting or deleting cell populations. (Hughes, 2007)