Measuring multiple apoptosis
parameters with the Agilent 2100
bioanalyzer
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A simplified and fast protocol for the analysis of DNA
fragmentation during apoptosis
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This Application Note describes a simplified and fast protocol for apoptosis
DNA fragmentation analysis with the Agilent 2100 bioanalyzer
using the DNA LabChip® kit. The combination of Annexin V, activecaspase-
3 and DNA laddering analysis on the same platform together
with the Cell Fluorescence LabChip kit permits quick confirmation and
quantitation of apoptosis in cell populations. A fast and simple protocol
was applied for the extraction of fragmented, chromosomal DNA. The
increased sensitivity and sizing accuracy of the DNA LabChip kit compared
to conventional agarose gel electrophoresis also means that a
significant lower amount of apoptotic cells is required for the analysis.
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The Agilent 2100 bioanalyzer was
introduced by Agilent Technologies
as the first commerially available
lab-on-a-chip analysis system
for the life science laboratory
using LabChip® products, developed
in collaboration with Caliper
Technologies Corp. Chip-based
approaches for a variety of separation-
based techniques have been
introduced, addressing DNA, RNA
and protein separations.1, 2, 3.
Recently the Agilent 2100 bioanalyzer
has been extended to enable
dual color simple flow cytometric
assays4. Apoptosis assays have
been demonstrated for quantitative
measurements of whole cells5.
The combination of cytometric
and gel-like separations on the
same instrument platform makes
it an ideal tool for multi-parametric
apoptosis quantitation and confirmation.
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Apoptosis
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| Apoptosis, or programmed cell
death, is the outcome of a metabolic
cascade that results in cells
dying in a controlled manner. It is
used by nature during development6,
homeostasis, aging and in
defense mechanisms. Apoptosis is
a key factor in cancer and also
suspected to be a characteristic
mechanism for degenerative disorders
such as Alzheimer7 or Parkinson's
diseases. The process is universal
and has been described in
eukaryotic cells as well as in individual
bacteria8, protozoa9 and
amoeba10. It is a target for research scientists in a wide variety
of fields. Apoptosis is characterized
by a distinct set of morphological
events involving plasma
membrane blebbing and asymmetry
loss, reduction of cell volume,
loss of mitochondrial membrane
potential, nuclear condensation,
fragmentation of DNA at
nucleosomal intervals and other
cytoplasmatic changes. Ultimately
fragmentation of the cell into
membrane-enclosed “apoptotic
bodies” occurs. Differential
expression patterns or alternate
mechanisms in the apoptosis pathways
among tissues and cell lines
makes it necessary to have two or
even three parallel experiments to
confirm, quantitate and compare
kinetic events on apoptosis. The appearance of DNA laddering
is unambiguously connected to
apoptosis. Targeting of phosphatidyl
serine (PS) in the outer
leaflet of cell membrane with fluorescently
labeled annexin V and
antibody labeling of active-caspase-
3 in cells has been previously
described in conjunction with the
Agilent 2100 bioanalyzer as a
quantitative measurement for
apoptotic cell samples5. |
This Application Note describes
the combined measurement of
annexin V and the extracted DNA
of apoptotic cells on one platform
– the Agilent 2100 bioanalyzer. A
fast protocol for DNA preparation
and recommendations for data
evaluation are discussed.
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Experimental
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Apoptosis was induced in Jurkat
cells by incubation in a 5 µM solution
of the topoisomerase
inhibitor Camptothecin (Sigma, #
C9911) in medium. Cells were harvested
and counted at the indicated
time intervals. Cell lysis and
DNA purification was performed
as described in the fast protocol
below. Eluted DNA was directly
loaded into the chip wells, prepared
according to the LabChip
Reagent Kit Guide.
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Fast protocol for DNA fragment
purification (based on original
protocol in reference 11)
• Pellet 1-2 × 106 cells by centrifugation
(2 min × 400 g).
Remove medium.
• Add 100 µL 4 °C lysis solution
(0. 2% Triton X, 10 mM Tris, 10
mM EDTA) and gently resuspend.
• Incubate 5 min at 4 °C.
• Centrifuge for 5 min at 13000 g.
• Transfer supernatant to new
vial and discard the pellet.
• Purify DNA using a low volume
elution method. Here a PCR
purification kit* was used
(QIAGEN MinElute PCR Purification
Kit. Cat.28004).
• Run in Agilent 2100 bioanalyzer
with the DNA 12000 LabChip
Kit, selecting the DNA 12000
Laddering assay from the biosizing
software.
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*As an alternative to the PCR
purification kit, the following protocol
has been successfully tested:
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• Add 2 µL 5M NaCl to the lysate
and vortex 5 seconds.
• Add 200 µL ice-cold absolute
ethanol and vortex 5 seconds.
• Incubate on ice for 10 minutes
• Centrifuge at 13000 g for 5 minutes.
Discard supernatant.
• Dry residual ethanol in speedvac
or desiccator.
• Resuspend precipitate in 30 µL
of pre-warmed (65 °C) distilled
water or 10 mM Tris/10 mM
EDTA.
• Run in Agilent 2100 bioanalyzer
with the DNA 12000 LabChip
kit, selecting the DNA 12000
laddering assay.
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UV measurements
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UV spectroscopy readings were
performed to determine total DNA
concentration. The Agilent 8453
UV-visible spectrophotometer was
used in conjunction with a 0.2-cm
light path ultra-micro cell (10 µl
volume). The whole spectrum
(190–1100 nm) was acquired and
evaluation was automatically done
by the Warburg-Christian
method12 with internal wavelength
correction at 320/10 nm.
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Annexin V apoptosis measurements
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A fast annexin protocol as
described in reference 13 was
used.
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Results and Discussion
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Besides DNA laddering, cytometric
assays like annexin V or caspase
analysis are commonly used
for apoptosis detection. Figure 1A
shows the analysis of annexin V in
Jurkat cells on the Agilent 2100
bioanalyzer after 6 hours treatment
with 5 µM campthotecin.
After switching the instrument to
electrophoresis mode, extracted
DNA from treated Jurkat cells was
analyzed using the DNA 12000
LabChip kit. The characteristic
DNA laddering further confirmed
apoptosis in the sample (figure
1B).
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Sizing information, provided by
the 2100 bioanalyzer software,
shows nucleosomal fragmentation
of DNA by evenly distributed
peaks in the electropherogram
(figure 2). Apoptosis is confirmed
in samples by fragmentation of the
chromosomal DNA in 180–200bp
intervals.14
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Typical DNA extraction with the
described fast protocol leads to
10 µL of 2-30ng DNA/µL (figure 3).
The DNA 12000 concentration
range specification is 0.5–50
ng/µL, so no further treatment is
required. Band concentration was
calculated to be approximately
1 ng/µL (1 µL loaded on the chip).
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Even at low concentrations, apoptotic
samples are clearly distinguishable
from the negative control
and necrotic samples (figure
4, samples 1 to 7). The positive
samples in figure 4 turned out to
be 35 % apoptotic as confirmed by
the annexin V assay. A clear discrimination
of samples that contain
a lower number of apoptotic
cells is achieved by overlaying
negative controls and samples in
the electropherogram view
(figure 5).
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If alternative DNA purification
methods are used, special attention
must be paid as the injection
of large amounts of genomic DNA
(like whole cell lysates) may lead
to clogging of the chip. The
recommended extraction protocol
eliminates such interference of
genomic DNA. In addition, fragmented
DNA is concentrated,
which permits the apoptosis measurement
of low cell numbers.
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Conclusion
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A combination of complementary
assays is usually required to confirm
that cells are apoptotic. The
2100 bioanalyzer offers the flexibility
to quantitatively measure
flow cytometric parameters and
subsequently perform a DNA fragmentation
assay. It eliminates the
need for outsourcing samples and
helps keep consolidated electronic
track of all results. In addition
to faster results and ease-of-use,
the high sensitivity and optimized
protocols allow minimum cell
consumption and saves sample
preparation time.
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