APOPTOSIS MEASUREMENT
Programmed cell death (apoptosis) is an active
process of cellular self-destruction. The difference with necrosis
is that it does not trigger tissue inflammation because the cell
membranes are not initially destroyed. The first characteristic
exploited in cytometry is that the cell undergoing apoptosis
undergoes a fragmentation of organelles and notably of the DNA of
the nucleus. These DNA fragments will eventually be released from
the cell in the apoptotic bodies.
Another characteristic of apoptotic cells that can be easily
exploited in cytometry is that they expose, on the outer leaflet
of the plasma membrane, phosphatidylserine which is normally
constitutive of the inner leaflet of the membrane.
All these characteristics can easily be used to study apoptosis by
cytometry.
DNA
fragmentation measurement.
As nuclei are
actively degraded, they lose part of their DNA content, with
fragments ending up in the apoptotic bodies. By facilitating the
exit of these apoptotic bodies from the cell and by marking the
DNA with a specific dye such as propidium iodide, cytometry can
detect on the one hand the cells that have not yet degraded their
DNA, which will therefore have their normal DNA content, and on
the other hand the cells that have lost their DNA fragments
(apoptotic cells), which will have a smaller quantity of DNA
(sub-G0/G1 peak). To detect these differences it is sufficient to
use a solution that facilitates the exit of apoptotic bodies from
the cell such as a solution (ethanol/water at 75/25% protocol
Cytobase). An example of this type of labeling is shown in Figure
1.
Figure 1 :
Detection of DNA fragmentation during apoptosis (sub G0/G1
peak in M1).
Measurement
with Annexin V
The apoptotic cell shows an early membrane
inversion towards phosphatidylserine which moves from the inner
side of the cell membrane when the cell is alive to the outer side
of the membrane when the cell enters apoptosis.
Annexin V is a protein with a high affinity for
phosphatidylserine, which could be used to perform early detection
tests of apoptosis. By coupling Annexin V to a fluorochrome, the
search for apoptotic cells by cytometry was greatly facilitated.
When testing the same cells as with the DNA fragmentation assay,
the measurement of the presence of phospatidylserines on the outer
membrane shows a higher percentage of Annexin V-labeled cells
because the detection of apoptosis is earlier with this technique
(Figure 2).
Figure 2 : Measurement of
apoptosis by Annexin V detection of phosphatidylserines on the
outer cell membrane
Multiparametric analysis of
apoptosis
Since apoptosis is an active phenomenon on living cells and living
cells have membrane integrity, it is possible to distinguish dead
cells from apoptotic cells by labeling with a DNA marker that does
not cross this plasma membrane (such as propidium iodide, 7AAD...).
It is possible to use multiparametric labeling to distinguish at the
same time cell viability and the presence of phosphatidylserine on
the outer membrane of the cell.
By using these two markers simultaneously it will be possible to
analyze more finely the state of the cells by distinguishing living
cells from early apoptotic cells and cells in necrosis/late
apoptosis (Figure 3).
Figure 3: Multiparametric
analysis of apoptosis by membrane expression of Annexin V (in
Y) and labeling of dead cells with 7AAD (in X).
