Apoptosis and Cell Culture Technology
237
population may represent fragments of apoptotic and necrotic nuclei, individual
apoptotic bodies containing various amounts of condensed chromatin and
individual chromosome or chromosomal aggregates from used mitotic cells.
Labelling of DNA strand breaks generated during DNA fragmentation can
be accomplished by labelling of the 3'OH termini at sites of DNA cleavage with
biotin conjegated nucleotides in a reaction catalysed by exogenous TdT [36, 37]
or DNA polymerase (nick translation) [38]. Gavrieli et al. [36] have given the
name 'TUNEL' which stands for 'Terminal transferase Utilizing Nick End
Labelling' for the above method of detecting apoptotic DNA strand breaks.
A kit called ApoptTag for the detection of apoptosis based on this technique is
now available from ONCOR. The method was later simplified by a single step
labelling DNA strand breaks with deoxynucleotides directly conjugated with
a fluorescent molecule, using exogenuous TdT [-39]. Using flow cytometry, this
method proved satisfactory for the detection of apoptosis in HL-60 and
lymphoma cultures.
There are several other flow cytometric techniques in use for assessing cell
viability. These techniques are based on the assessment of the structural and
functional integrity of the plasma membrane using, for example, the exclusion of
propidium iodide (PI) and cellular retention of fluorescin diacetate. The exclusion of PI is also used in combination with uptake of the cationic flurochrome
Rh123, normally used to study mitochondrial activity. Live cells with active
mitochondria exclude PI and retain Rh123. While these methods are adequate
to identify necrotic cells they cannot discriminate between viable and apoptotic
cells nor between necrotic and secondary necrotic cells. Generally they can be
functionally considered as similar to the non-flurochrome based cytotoxicity
assays such as trypan blue exclusion, release of 51Cr or LDH from damaged
cells, uptake of neutral red into lysosomes and conversion of MTT to formazan
via the mitochondrial dehydrogenase activity.
Finally, it is important to recognise that although flow cytometry is quantitative, reproducible and can be applied to a variety of cell types, the observed
morphological characteristics of cells undergoing apoptosis vary not only between cell types, but also between different triggers of apoptosis and can be
absent or misleading in some cases.
4 Mechanisms and Death Signals
The activation of the apoptotic pathways invovles a diverse variety of physiological and non-physiological triggers determined by the presence or absense
of extracellular signals. Examples of the signals which trigger apoptosis are
y-irradiation [40], glucocorticoides [41], interaction with a surface receptor, e.g.
Fas or APO-1 [-42] and removal of essential growth factors and/or survival
factors [e.g. 43], (see Table 4).
237
population may represent fragments of apoptotic and necrotic nuclei, individual
apoptotic bodies containing various amounts of condensed chromatin and
individual chromosome or chromosomal aggregates from used mitotic cells.
Labelling of DNA strand breaks generated during DNA fragmentation can
be accomplished by labelling of the 3'OH termini at sites of DNA cleavage with
biotin conjegated nucleotides in a reaction catalysed by exogenous TdT [36, 37]
or DNA polymerase (nick translation) [38]. Gavrieli et al. [36] have given the
name 'TUNEL' which stands for 'Terminal transferase Utilizing Nick End
Labelling' for the above method of detecting apoptotic DNA strand breaks.
A kit called ApoptTag for the detection of apoptosis based on this technique is
now available from ONCOR. The method was later simplified by a single step
labelling DNA strand breaks with deoxynucleotides directly conjugated with
a fluorescent molecule, using exogenuous TdT [-39]. Using flow cytometry, this
method proved satisfactory for the detection of apoptosis in HL-60 and
lymphoma cultures.
There are several other flow cytometric techniques in use for assessing cell
viability. These techniques are based on the assessment of the structural and
functional integrity of the plasma membrane using, for example, the exclusion of
propidium iodide (PI) and cellular retention of fluorescin diacetate. The exclusion of PI is also used in combination with uptake of the cationic flurochrome
Rh123, normally used to study mitochondrial activity. Live cells with active
mitochondria exclude PI and retain Rh123. While these methods are adequate
to identify necrotic cells they cannot discriminate between viable and apoptotic
cells nor between necrotic and secondary necrotic cells. Generally they can be
functionally considered as similar to the non-flurochrome based cytotoxicity
assays such as trypan blue exclusion, release of 51Cr or LDH from damaged
cells, uptake of neutral red into lysosomes and conversion of MTT to formazan
via the mitochondrial dehydrogenase activity.
Finally, it is important to recognise that although flow cytometry is quantitative, reproducible and can be applied to a variety of cell types, the observed
morphological characteristics of cells undergoing apoptosis vary not only between cell types, but also between different triggers of apoptosis and can be
absent or misleading in some cases.
4 Mechanisms and Death Signals
The activation of the apoptotic pathways invovles a diverse variety of physiological and non-physiological triggers determined by the presence or absense
of extracellular signals. Examples of the signals which trigger apoptosis are
y-irradiation [40], glucocorticoides [41], interaction with a surface receptor, e.g.
Fas or APO-1 [-42] and removal of essential growth factors and/or survival
factors [e.g. 43], (see Table 4).