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Mohamed A1-Rubeai
3.2.1 Microscopically Based Methods
The low incidence of the occurrence of apoptotic cells in tissues and their rapid
disappearance due to phagocytosis makes them difficult to detect. In contrast, in
vitro cell culture provides a process for significantly increasing the number of
spontaneous or induced transient apoptotic cells. Microscopy can be employed
to detect and examine the morphology of apoptotic cells either indirectly after
processing and staining of a section or directly by in situ examination of
unstained cells. Evan and co-workers used time-lapse video microscopy to
monitor and study apoptotic events induced by c-myc in serum-deprived cells
[25]. However, stains such as toluidine blue, acridine orange and silver stain
have been used to demonstrate the condensation of chromatin [21, 26-28]. We
use dual staining with acridine orange and propidium iodide of unfixed cells to
differentiate between necrotic, apoptotic and viable cells (Fig. 4b). The technique
is employed to obtain quantitative information on the kinetics of cell death in
various commercially important cell lines and to assess the influence of medium
components and the physical environment on cell growth and death in bioreactors. It is worth noting that this technique can recognise those cells which have
undergone apoptosis and subsequently lost their membrane integrity to become
'secondary necrotic' as well as both types of apoptotic bodies- with or without
chromatin. The clearest way to demonstrate the morphological characteristics
of apoptosis remains electron microscopy (Fig. 4a). Although this technique is
not suitable for quantitation of apoptosis, it can illustrate detailed morphological features of the process, such as the state of cytoplasmic organelles, cellular
membrane and nuclear structure. A1-Rubeai et al. [29] demonstrated the occurrence of apoptosis in hybridoma cell culture and its ultrastructural features by
transmission and scanning electron microscopy. Their work indicated that the
cell membrane undergoes structural changes during the late stages of batch
culture with the disappearance of microvilli and the appearance of blebs and
deep indentations. Further ultrastructural features of apoptosis has been shown
to include very late cytoplasmic damage despite the good preservation of the
plasma membrane and cytoplasm [-30].
3.2.2 DNA Fragments
DNA fragmentation into oligonucleosome-size fragments occurs simultaneously with the manifestation of morphological changes such as chromatin
condensation. The oligonucleosomal fragments can be visualized on electrophoretic agarose DNA gel as a distinctive ladder of multiples of 180-200 bp
(Fig. 5). In contrast to apoptosis, DNA fragments produced during necrosis are
of irregular sizes, resulting in a smear on agarose gel. Although the ladder
pattern of DNA fragments has been considered as the biochemical hallmark of
apoptosis, in many cases the classical morphological characteristics of apoptosis
can be manifested in the absence of DNA fragmentation [31, 32]. Moreover, it is
Mohamed A1-Rubeai
3.2.1 Microscopically Based Methods
The low incidence of the occurrence of apoptotic cells in tissues and their rapid
disappearance due to phagocytosis makes them difficult to detect. In contrast, in
vitro cell culture provides a process for significantly increasing the number of
spontaneous or induced transient apoptotic cells. Microscopy can be employed
to detect and examine the morphology of apoptotic cells either indirectly after
processing and staining of a section or directly by in situ examination of
unstained cells. Evan and co-workers used time-lapse video microscopy to
monitor and study apoptotic events induced by c-myc in serum-deprived cells
[25]. However, stains such as toluidine blue, acridine orange and silver stain
have been used to demonstrate the condensation of chromatin [21, 26-28]. We
use dual staining with acridine orange and propidium iodide of unfixed cells to
differentiate between necrotic, apoptotic and viable cells (Fig. 4b). The technique
is employed to obtain quantitative information on the kinetics of cell death in
various commercially important cell lines and to assess the influence of medium
components and the physical environment on cell growth and death in bioreactors. It is worth noting that this technique can recognise those cells which have
undergone apoptosis and subsequently lost their membrane integrity to become
'secondary necrotic' as well as both types of apoptotic bodies- with or without
chromatin. The clearest way to demonstrate the morphological characteristics
of apoptosis remains electron microscopy (Fig. 4a). Although this technique is
not suitable for quantitation of apoptosis, it can illustrate detailed morphological features of the process, such as the state of cytoplasmic organelles, cellular
membrane and nuclear structure. A1-Rubeai et al. [29] demonstrated the occurrence of apoptosis in hybridoma cell culture and its ultrastructural features by
transmission and scanning electron microscopy. Their work indicated that the
cell membrane undergoes structural changes during the late stages of batch
culture with the disappearance of microvilli and the appearance of blebs and
deep indentations. Further ultrastructural features of apoptosis has been shown
to include very late cytoplasmic damage despite the good preservation of the
plasma membrane and cytoplasm [-30].
3.2.2 DNA Fragments
DNA fragmentation into oligonucleosome-size fragments occurs simultaneously with the manifestation of morphological changes such as chromatin
condensation. The oligonucleosomal fragments can be visualized on electrophoretic agarose DNA gel as a distinctive ladder of multiples of 180-200 bp
(Fig. 5). In contrast to apoptosis, DNA fragments produced during necrosis are
of irregular sizes, resulting in a smear on agarose gel. Although the ladder
pattern of DNA fragments has been considered as the biochemical hallmark of
apoptosis, in many cases the classical morphological characteristics of apoptosis
can be manifested in the absence of DNA fragmentation [31, 32]. Moreover, it is