Theoretical and practical background about an Apoptosis Assay

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Theoretical and practical background about
anApoptosis Assay

• Dr. Adel RA Abd-Allah

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Apoptosis is a programmed cell death that occurs
in different tissues. It has been shown to pla
y a key role in the normal development of
different types of cells particularly in the
immune system Its hallmark biochemical feature
is endonuclease activation, giving rise to
internucleosomal DNA fragmentation
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There are also characteristic morphological
changes, including chromatin condensation, nuc
lear fragmentation and shrinkage as well as
formation of dense chromatin masses (apoptotic
bodies). In apoptosis, nuclear changes are obs
erved first, in contrast to the changes seen in
necrosis, which usually begins with cell membrane
damage. For these differences, apoptotic cells
exclude trypan blue dye until late in the
process to look viable, whereas necrotic cells
take up the dye early and look dead under light
microscope.
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Because DNA fragmentation in apoptosis is an
enzymatic process (internucleosomal),
characteristic DNA ladders or pattern can be
visualized on gel electrophoresis as a series of
fragments with lengths which are equal to or
multiples of 180-200 base pair
In the contrary, the random DNA cleavage in case
of necrosis leads to a DNA smear on gel
electrophoresis. Apoptosis can be initiated by
toxicants, drugs or perturbation of the cellular
homeostasis in some disease conditions.
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Apoptosis in target cells can be evaluated by
detecting the pattern of DNA fragmentation on
agarose gel electrophoresis. The percentage of
DNA fragmentation can also be assessed according
to the procedure of Perandones et al., (1993).
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Procedure
1- Cells (10x106 cells) are pelleted by
centrifugation (1500 rpm for 5 min),
resuspended in 400 µl hypotonic lysis
buffer (10 mM Tris, 0.2 Triton X-100 and 1mM
EDTA, pH 8.0) and centrifuged for 15 min
at 13800 xg. 2- The supernatants (SN) containi
ng small DNA fragments were separated one-half
of the volume was used for gel electrophoresis
while the other half, together with the pellet
containing large pieces of DNA, were used to
quantify the fragmented DNA via Diphenylamine
(DPA) assay.
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3-The samples were treated with equal volumes of
absolute isopropyl alcohol and 0.5 M NaCl in
order to precipitate DNA. The samples were then
kept at 20C overnight and centrifuged at 13800
xg for 15 min. 4-The pellets were then washed
with 200 µl of 70 ethanol and allowed to dry at
room temperature. 5-The extracted DNA was reco
nstituted in 12 µl of Tris-EDTA buffer (10 mM
Tris-Hcl , 1 mM EDTA, pH 7.4), and 3 µl looding
buffer 50 glycerol, 1 x TAE (Tris acetate
EDTA), 10 saturated bromophenol blue, and 1
xylene cyanol.
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6-The samples are incubated at 37C for 20 min,
and then electrophoresed on 1 agarose gels
containing 0.71 µg/ml ethidium bromide. At the
end of the runs, gels are examined using UV
transillumination.
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DPA procedure 1-The DPA assay reaction was mod
ified by Perandones et al.,(1993) from
Burton(1956). Briefly, perchloric acid (0.5 M)
was added to the pellets containing native DNA
(reconstituted in 200 µl of the hypotonic lysis
buffer) and to the supernatants containing
fragmented DNA followed by the addition of 2
volumes of a solution containing 0.088 M DPA, 98
V/V glacial acetic acid, 1.5 V/V concentrated
sulfuric acid and 0.5 acetaldehyde solution.
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The samples are kept at 4C for 48 h. The
colorimetric reaction is then measured
spectrophotometrically at 575 nm. The percentage
of DNA fragmentation can be expressed by the
formula Percent of DNA fragmentation OD575
supernatant X 100 2 X (OD575 OD575 pellet
s)
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References Perandones CE, Illera VA, Peckham D
, Stunz LL, Ashman RF.Regulation of apoptosis in
vitro in mature murine spleen T cells.J Immunol.
1993151(7)3521-9.
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