Identification of Apoptosis

The search for simple techniques that allow for the identification of apoptotic cells has attracted a considerable

Apoptosis Diagram

Figure 1. Schematic diagram showing the morphological features of apoptotic and necrotic cell death. The cell shown at stage 1 of necrosis exhibits clear swelling and dilation of mitochondria and Golgi. At this stage, nuclear morphology remains unchanged. By stage 2 of necrosis, disruption of organelles and plasma membrane is apparent, nuclear morphology is altered, and complete destruction of the cell quickly follows. During stage 1 of apoptosis, on the other hand, the cell exhibits extensive boiling or blebbing of the plasma membrane and a reduction in cellular volume. Condensation of the nuclear morphology may also be apparent. By stage 2, the chromatin has condensed and fragmented into several spherical particles and the cell is smooth surfaced in appearance. Apoptotic bodies containing chromatin and organelles are also shown. Eventually, plasma-membrane integrity is lost and degraded chromatin escapes from the dead cells, leaving nuclear-free ghosts. These structures may persist in the culture for many days.

Figure 1. Schematic diagram showing the morphological features of apoptotic and necrotic cell death. The cell shown at stage 1 of necrosis exhibits clear swelling and dilation of mitochondria and Golgi. At this stage, nuclear morphology remains unchanged. By stage 2 of necrosis, disruption of organelles and plasma membrane is apparent, nuclear morphology is altered, and complete destruction of the cell quickly follows. During stage 1 of apoptosis, on the other hand, the cell exhibits extensive boiling or blebbing of the plasma membrane and a reduction in cellular volume. Condensation of the nuclear morphology may also be apparent. By stage 2, the chromatin has condensed and fragmented into several spherical particles and the cell is smooth surfaced in appearance. Apoptotic bodies containing chromatin and organelles are also shown. Eventually, plasma-membrane integrity is lost and degraded chromatin escapes from the dead cells, leaving nuclear-free ghosts. These structures may persist in the culture for many days.

amount of interest. As already stated, visualization of nuclease-mediated cleavage of DNA has been widely used to identify the presence of apoptotic cells. However, the technique can produce variable quality results that are of a qualitative, rather than a quantitative, nature.

Perhaps the simplest techniques for the study of apop-tosis are based on the identification of the morphological features of cell death. For example, fluorescence microscopic analysis of nuclear morphology is a highly effective method for identification and quantification of apoptosis. There are two ways in which this may be done. If samples cannot be analyzed immediately, cells may be fixed in formaldehyde and stored at 4 °C. Analysis involves staining with acridine orange, which reveals the condensation of chromatin in apoptotic cells (7). However, this technique has one major drawback—an inexperienced operator may confuse early necrotic cells with viable cells, which have a very similar nuclear morphology. In order to avoid this difficulty, cells may be analyzed immediately while still in their culture medium by using the acridine orange-propidium iodide dual-staining technique (8). All cells are permeable to acridine orange, which stains chromatin green. Only membrane-damaged cells take up propidium iodide, and as a result exhibit red fluorescence. This technique therefore provides information regarding plasma membrane integrity, as well as nuclear morphology, and can consequently be used to simplify identification of ne-crotic cells. Additionally, the classification ofapoptotic cells into early apoptotic and membrane-damaged apoptotic (sometimes referred to as secondary necrotic) cells gives an indication of the cell growth and death kinetics under the influence of a variety of environmental conditions during the cultivation process. The early-membrane-intact phase of death is relatively brief and, therefore, the presence of a large proportion of cells at this stage indicates that the rate of cell death is very high.

Microscopic techniques have two major drawbacks— they are subjective and time consuming. In theory, the development of flow cytometric methods should overcome these difficulties and provide a powerful tool for the study of the biochemical features of apoptosis in heterogenous cell populations. At present, there are a number of techniques that are available, and some of the most commonly used are discussed further.

Light-scattering Properties. The simplest flow cytometric method is based on the changes in light scattering properties that accompany cell death. Cells can be studied without the need for pretreatment, with the decrease in cell size producing a decrease in forward-scattered light. The increased granularity caused by nuclear condensation produces an increase in orthogonal light scatter (9,10). However, the latter is a transient stage, and eventually, a reduction in orthoganol light scatter is observed. The technique can also be used for the identification ofnecrotic cells (at least in their early stages). When necrosis is induced by a permeablizing agent such as saponin, a reduction in forward scatter is observed, but the increase in side scatter that occurs during apoptosis is not seen.

Changes in DNA Content. When stained with a DNA-specific stain such as propidium iodide (PI), apoptotic cells exhibit a characteristically low DNA content that appears as a sub-G1 peak (i.e., it appears below the position of the G1 peak of the cell cycle of viable cells). This is believed to result from the leakage ofcleaved DNA from apoptotic cells (11-14). The technique provides a rather good correlation with the fluorescence microscopic technique already described. However, necrotic cells undergoing degradation may also exhibit a reduced DNA content, although the passive and asynchronous nature of this process means that a clear "peak" is not usually observed.

Annexin V. In viable cells, phosphatidylserine (PS) is located on the inner leaflet of the plasma membrane (15). During apoptosis, one of the earliest changes is the loss of this asymmetrical distribution (15,16). Annexin V has a very high affinity for PS. Conjugation of annexin V to a fluorescent tag, such as FITC, enables the use of this interaction to identify cells that have lost PS asymmetry. By combining this method with PI staining, it is also possible to classify apoptotic cells into two subpopulations: early (membrane intact and therefore PI negative) and late (membrane damaged and therefore PI-positive). The technique has been found to give a good correlation with levels of apoptosis during hybridoma batch cultures, during which apoptosis accounts for around 90% of cell deaths, as revealed by the fluorescence microscopic method described earlier. However, as with other flow cytometric techniques, necrotic cells can also give a false-positive result. This is because damage to the plasma membrane allows the annexin to enter the dead cell and bind to PS residues on the inner surface of the plasma membrane. Thus, the technique is reliable only when used to analyze early apoptotic cells, which can be seen as Annexin V positive and PI negative (16,17).

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Responses

  • brad russell
    What kind of apoptosis are there in cell culture?
    6 years ago

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