Comparison of Several Techniques for the Detection of Apoptotic Cells

Apoptosis or programmed cell death is a specific cellular event with distinct morphological, histological, molecular properties and biochemical mechanisms. It plays an important role in the normal turnover, development, and function of cells.

Consequently, the analysis of apoptosis is of interest in many basic research and clinically oriented investigations. A number of methods have been developed to detect apoptosis. These methods are based on the changes in different apoptotic events, including plasma membrane alterations, mitochondrial changes, changes in the cytoplasm, DNA modifications and so on.

Morphology

The apoptotic process involves a sequence of cell shrinkage, increased cytoplasmic density, chromatin compaction, and segregation into sharply circumscribed masses that abut on the nuclear membrane and can form blister-like protrusions. The latter then separate to produce membrane-bound apoptotic bodies.

These morphological changes can be detected using the electron microscope. The application of quantitative digital imaging techniques to electron microscopy enables the detection of even subtle changes in nuclear densities. Membrane blebbing and apoptotic bodies can also be seen by simple phase-contrast light microscopy. Another versatile tool is confocal laser scanning microscopy, powerful for both morphological analysis and macromolecular localization.

• Advantages

1) It is a relatively reliable and inexpensive method for detecting apoptotic cells
2) The plethora of information provided is wide, may be useful for subsequent biochemical or molecular studies, furnishing important controls for the experiment

• Disadvantages

1) Quantitative measurement lacks objectivity and reproducibility
2) The procedure is time consuming and is fairly expensive
3) Many samples cannot be analyzed, it requires laborious preparation
4) Because only a small area can be visualized, quantification of the extent of apoptosis is also difficult

Plasma Membrane Alterations

Plasma membrane changes are one of the first characteristic features of apoptosis. In the early stages of apoptosis, the lipid phosphatidylserins translocate from the inner to the outer surface of the plasma membrane.

Annexin V is a calcium-dependent protein that preferentially binds phosphatidylserine with high affinity, which can be used to rapidly detect this early cell surface marker of apoptosis. Annexin V used in conjunction with DNA binding dyes can be used to distinguish between apoptotic cells (single staining) and necrotic (double staining) cells.

• Advantages

1) Easy, rapid and accurate quantitation of apoptosis in both viable and fixed single cells.
2) Explain the relationship between induction of apoptosis by different agents and their cell cycle phase specificity.

• Disadvantages

1) Very time consuming as it has multiple steps and is quantitative. Therefore, intact tissues usually require pre-treatment with an enzyme to release the individual cells for analysis.

Changes in the Cytoplasm

The most significant change in the cytoplasm during apoptosis is the activation of caspases. Once activated, they form dimers and activate other caspases. This process is known as the caspase cascade.

Caspases are synthesized as inactive precursors and activated as a consequence of signaling induced by a wide range of physiological and pathological stimuli. These proteases cleave a diverse group of intracellular substrates, contributing to various manifestations of apoptosis. Some caspases (e.g. 2, 8, 9, and 10) initiate apoptosis, and others (e.g. 3, 6, and 7) are downstream effectors.

• Advantages

1) Detection of active caspase 3 in situ is an easy, sensitive, and reliable method for quantifying apoptosis.
2) Caspase activation can be detected in a variety of ways including western blot, immunoprecipitation and immunohistochemistry.
3) This technique allows selection for individual initiator or execution caspases. It also allows for rapid and consistent quantification of apoptotic cells.

• Disadvantages

1) The integrity of the sample is destroyed thereby eliminating the possibility of localizing the apoptotic event within the tissue or determining the type of cell that is undergoing apoptosis.
2) Caspase activation does not necessarily indicate that apoptosis will occur.
3) Moreover, there is tremendous overlap in the substrate preferences of the members of the caspase family, affecting the specificity of the assay.

DNA Modifications

Chromatin condensation and DNA fragmentation are one of the hallmarks of apoptosis. At the late stage of apoptosis, caspase-activated endonucleases cause double-stranded DNA breaks. These apoptotic nucleosome fragments (180-200 bp) can be resolved by gel electrophoresis as typical DNA ladders.

The terminal deoxy-nucleotidyl transferase mediated dUTP nick end labelling (TUNEL) assay utilizes TdT to mark those breakpoints with tagged (e.g. biotinylated) nucleotides, which are then detected using enzyme-tagged (for IHC) or fluorescently labelled (for FACS) antibodies. TUNEL indicates DNA cleavage from any form of cell death, necrotic cells may also be labelled.

• Advantages

1) The reactions are based on the direct labelling of 3′ -hydroxyl termini of DNA breaks, and thus the lesions measured are identifiable at the molecular level.
2) The DNA breaks occur very early in apoptosis, prior to changes in cell morphology, therefore, these assays can be applied to study the very early events of apoptosis.
3) Precise in the determination of cell death and DNA damage.
4) DNA ladder assay and TUNNEL staining have high sensitivity.

• Disadvantages

1) Provide qualitative rather than quantitative results.
2) These procedures have multiple steps and require more time.
3) The sensitivities and specificities of these techniques depend on fixative used, pre-treatment and concentration of terminal transferase enzyme.

References

1. Sgonc R. et al.; Apoptosis detection: an overview. Experimental Gerontology, 1998, 33(6): 525-533.
2. Shwetha Nambiar K. et al.; Apoptosis detection modalities: a brief review. International Dental & Medical Journal of Advanced Research, 2016, 2: 1-5.
3. Sgonc R. et al.; Methods for the detection of apoptosis. Int Arch Allergy Immunol, 1994, 105: 327-332.

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