Apoptosis detection by annexin V and
active caspase-3 with the Agilent 2100
bioanalyzer
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This Application Note describes how the Agilent 2100 bioanalyzer can
be used to analyze apoptotic cell samples. Performance of two assays
for apoptosis detection is demonstrated and the data are compared to
those obtained with a conventional flow cytometer. Histogram quality
of the low number of fluorescent cells counted with the microfluidic
sytem is in good agreement with data obtained with the flow cytometer.
Detailed protocols and reagent recommendations for an annexin V
assay and a caspase-3 assay are given. High reproducibility of chip
results, low cell consumption and ease-of-use are advantages the compact
Agilent 2100 bioanalyzer system offers for apoptosis cell assays.
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The Agilent 2100 bioanalyzer was
introduced by Agilent Technologies
as the first commercially
available lab-on-a-chip analysis
system for the life science laboratory
using LabChip® products,
developed by Caliper Technologies
Corp. Chip-based approaches
for a variety of separation-based
techniques have been introduced,
addressing DNA, RNA, and protein
separations. The Agilent 2100 bioanalyzer
is capable of two-color
fluorescence detection and runs
disposable microfluidic glass
chips. The applications presented
here are based on the controlled
movement of cells by pressure-driven
flow inside the interconnected
networks of microfluidic channels.
Cells are hydrodynamically
focused in these channels before
passing the fluorescence detector
in single file. Each chip accommodates
up to six samples and data
acquisition of all samples is fully
automated while data analysis
allows for user-specific settings.
Specific advantages of the instrument
are the low number of cells
required for analysis and the easeof-
use. Here, the detection
of apoptotic cell death is investigated.
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Apoptosis
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Apoptosis, or programmed cell
death, is a genetically controlled
response of cells to commit suicide.
The purpose of this process
is to kill unwanted host cells. It is
used in three situations: for development
and homeostasis, as a
defense mechanism and in aging.
Apoptosis is characterized by a distinct set of morphological
events involving plasma membrane
blebbing, loss of cell volume,
nuclear condensation, fragmentation
of DNA at nucleosomal
intervals and ultimate fragmentation
of the cell into membraneenclosed
“apoptotic bodies”1.
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During the early phase of apoptosis,
changes occur at the plasma
membrane. Phosphatidyl serine
(PS) that is actively confined to
the inner leaflet of the lipid bilayer
in healthy cells translocates to the
outer layer, where it is exposed at
the external surface of the cell2.
Annexin V belongs to the family of
calcium and phospholipid binding
proteins with high affinity for PS3.
It can be used as a sensitive probe
for PS exposure on the cell membrane.
Translocation of PS to the
external cell surface is not unique
to apoptosis, as it also appears
during necrosis. These mechanisms
of cell death differ in their
initial stages when the cell membrane
remains intact during apoptosis
but loses integrity, leaking
cellular contents, during necrosis4.
Therefore, measuring annexin V
binding to the cell surface as indicator
for apoptosis has to be performed
in conjunction with a calcein
retention test that verifies the
integrity of the cell membrane. As
electrically neutral or nearly neutral
molecules, calcein acetyloxymethyl
(AM) esters freely diffuse
into most cells. Once inside the
cell, these non-fluorescent substrates
are converted by nonspecific
intracellular esterases into
fluorescent products that are
retained by cells with intact plasma
membranes. In contrast, both
the unhydrolyzed substrates and their products rapidly leak from
dead or damaged cells with compromised
membranes, even when
the cells retain some residual
esterase activity5.
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Apoptosis involves active participation
of endogenous cellular
enzymes in the mediation of death
well before membranes lose their
integrity. A family of cysteine proteases
(caspases) appears to
represent the effector arm of the
apoptotic program6. Caspase activation
can be achieved by different
pathways, by the cell surface
death receptor pathway and by
the mitochondria-initiated pathway.
In the cell surface death
receptor pathway, activation of
caspase-8 following its recruitment
to the death-inducing signaling
complex (DISC) is the critical
event that transmits the death signal.
This event is regulated at several
different levels by various
viral and mammalian proteins.
Activated caspase-8 can activate
downstream caspases by direct
cleavage or indirectly by inducing
cytochrome c release from the
mitochondria. In the mitochondrial-
initiated pathway, caspase activation
is triggered by the formation
of a multimeric Apaf-1/cytochrome
c complex that is fully
functional in recruiting and activating
procaspase-9. Activated
caspase-9 then cleaves and activates
downstream caspases such
as caspase-3, -6, and -77. Caspase-3
is a key protease that is activated
during the early stages of apoptosis
and, like other members of the
caspase family, is synthesized as
an inactive proenzyme that is
processed in cells undergoing
apoptosis by self-proteolysis and/or cleavage by another protease8.
Active caspase-3, a marker
for cells undergoing apoptosis,
consists of a heterodimer of 17
and 12 kDa subunits which is
derived from the 32 kDa proenzyme9.
A convenient and broadly
accepted method to study the
activity of caspase-3 during apoptosis
involves intracellular antigen
staining with anti-active caspase-3
antibodies. Following fixation,
permeabilization and staining, the
activity of caspase-3 can be measured
by flow cytometry or fluorescence
microscopy techniques.
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Here, we describe the measurement
of percentages of apoptotic
cells within cell populations using
the Agilent 2100 bioanalyzer. The
apoptotic process was induced in
Jurkat cells, a cell line derived
from an acute human T-cell leukemia, either by treatment with
camptothecin or by incubation
with anti Fas-antibodies. Different
readouts were chosen – either
annexin-presenting cells within the
live cell population were detected
or the percentage of cells with
active caspase-3 was measured in
the total cell population.
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Experimental
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The Agilent 2100 bioanalyzer
and Cell Assay Extension were
obtained from Agilent Technologies
Deutschland GmbH (Waldbronn,
Germany). Detection of
apoptotic cells was performed on
the Agilent 2100 bioanalyzer in
combination with the Cell Fluorescence
LabChip® Kit and the Cell
Fluorescence software. The kit
includes 25 chips and reagents
required to perform the analysis of pre-stained cells. Stained cell samples
were resuspended in an
isobuoyant cell buffer at 2 x 106
cells/ml and loaded onto the chips
as described in the reagent kit
guide. Data acquisition was performed
using an intuitive software
package with no requirement to
manually set instrument specific
parameters.
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Results
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a) Induction of apoptosis with
camptothecin; annexin V staining
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In a first application example,
Jurkat cells were induced with
camptothecin and harvested after
0, 4 and 16 hours of treatment.
After washing, the cells were incubated
with annexin V-biotin. Cells
were washed again and stained
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with Cy5®-streptavidin and calcein
according to protocol. After
washing and resuspending the
cells in cell buffer the samples
were loaded onto the chip and
inserted into the Agilent 2100 bioanalyzer.
A predefined assay was
chosen from the system software
for chip measurement. Figure 1
shows representative histograms
and dot plots of an untreated control
sample and a sample treated
for 16 hours. The sub-populations
of all living cells were defined by
setting a marker for all calcein
stained cells (figure 1 A+D). These
sub-populations are further analyzed
in the red color by setting a
marker for all cells showing a significant
red staining by annexin-V
(figure 1 B+E). In this way, all
cells that show strong annxin-V
binding and a strong calcein signal,
indicating an intact cell membrane,
are detected. These apoptotic
cells can also be seen in a
dot plot view as shown in the rectangular
areas in figure 1 C and 1
F. The data quality is comparable
to the data quality generated with
the same samples on a conventional
flow cytometer (figure 2),
which usually counts many more
cell events for analysis. Final
results are expressed as percentage
apoptotic cells of all live cells.
Figure 3 shows the results which
demonstrate a good reproducibility
of the measurement over several
chips. The figure also shows
data from a conventional flow
cytometer which nicely compares
with the data obtained with the
Agilent 2100 bioanalyzer.
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b) Induction of apoptosis with Fas
antibody; caspase-3 staining
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In a second application example
Jurkat cells were treated for two
and four hours with anti Fas antibodies
to induce apoptosis. In this
application a different detection
method for apoptosis, intracellular
staining of active caspase-3 is used.
After washing half of the cells were
stained with SYTO16 (an intercalating
nucleic acid stain), washed
again and treated with a permeabilization/
fixation solution. They
were subsequently stained with
anti active caspase-3 antibodies
and a Cy5- conjugated secondary
antibody (figure 4 A). The other
half of the treated cells were
stained with calcein/annexin V-Cy5
as described above (figure 4 B).
Both sample groups were measured
on the 2100 bioanalyzer and
on a conventional flow cytometer.
Only 20,000 cells of each prepared
cell sample were loaded onto the
chip (500-1000 cells analyzed per
sample), whereas 100,000 cells
were required for measurement on
the flow cytometer (10,000 cells
analyzed per sample). As expected,
the percentages of live apoptotic
cells are similar for both assays.
The Agilent 2100 bioanalyzer displayed
good reproducibility and
yielded data comparable to the
flow cytometer system.
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Materials and methods
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Cells were cultured in RPMI medium
containing 10 % fetal calf
serum, penicillin/streptomycine, 1
mM Sodium Pyruvate and 2 mM Lglutamine.
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a) Induction of apoptosis and
annexin V assay
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Reagents
• Camptothecin: Prepare a
10 mM solution in DMSO.
• Annexin V-Biotin Apoptosis
Detection Kit: Includes annexin
V-biotin, 5x binding buffer and
media binding reagent
• Fluorolink Cy5 labeled
streptavidin: Reconstitute with
1 ml distilled water to yield a
concentration of 1 mg/ml
• Paraformaldehyde 16 % solution
EM Grade (optional)
• Calcein-AM: Dilute the
original stock with DMSO to
yield a 500 µM solution.
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Protocol for annexin V assay
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1. Treat Jurkat cells at a
concentration of 5 x 105/ml
with 1 µM camptothecin or
0.5 µg/ml anti Fas-antibody for
2–24 h to induce apoptosis.
2. Harvest control and camptothecin-
treated or Fas antibody-
treated cells and adjust
cell density to 1 x 106 cells /ml
in culture medium.
3. Add 10 µl media binding
reagent per 500 µl of cell suspension
(5 x 105 cells).
4. Add 1.25 µl annexin V-biotin
per 500 µl of cell suspension
(5 x 105 cells). Incubate for 15
minutes at room temperature
(RT).
5. Centrifuge cells at 400x g for
4 minutes. Remove medium and resuspend cells at a cell density
of 1 x 106 cells /ml in
1x binding buffer. Use 5x binding
buffer and dilute to 1x with
distilled water.
6. Add 1 µl Cy5-streptavidin and
0.5 µl calcein (calcein stock =
500 µM, final = 0.5 µM) per
500 µl of cell suspension (5 x 105
cells). Incubate in the dark for
10 minutes at RT.
7. Wash cells once with 500 µl of
1 x binding buffer.
8. Optional step if cells are to be
be stored overnight and
measured later:
Remove medium and resuspend
cells well at a cell density of 1 x
106 cells /ml in 1x binding buffer.
Make sure there are no cell
clumps in the suspension. Add
70 µl paraformaldehyde 16 %
while stirring. Incubate for 10
minutes at RT and store the
cells at 4 °C.
9. Resuspend cells in cell buffer
at 2 x 106 cells/ml and load on
chip according to the reagent kit
guide.
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b) Induction of apoptosis and
caspase 3 assay
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Reagents
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• BD FACS Permeabilizing
Solution 2 (500 tests)
• Camptothecin: Prepare a
10 mM solution in DMSO.
• Rabbit anti-active caspase-3 mAb
• Cy5-conjugated AffiniPure
F(ab')2 fragment goat anti-rabbit
IgG (H+L)
• SYTO16
• Staining buffer (PBS, 2 % BSA,
0.05 % NaN3)
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Protocol for caspase 3 assay
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1. Treat 5 x 105/ml Jurkat cells
with 1 µM camptothecin or
0.5 µg/ml anti Fas antibody for
2–24 hours to induce apoptosis.
2. Harvest control and camptothecin-
treated or Fas antibody-
treated cells and adjust
cell density to 1 x 106 cells /ml
in staining buffer.
3. Add 1.5 µl SYTO16 per ml and
incubate 10 minutes at 37 °C.
4. Wash cells in 2 ml staining
buffer and centrifuge (500 x g, 5
minutes).
5. Resuspend 5 x 105 cells in
500 µl 1x BD permeabilizing
solution (dilute in distilled
water) and incubate 10 minutes
at RT.
6. Wash cells in 2 ml staining
buffer and centrifuge (500 x g,
5 minutes).
7. Add anti caspase-3 antibody at
5 µg/ml in a total of 100 µl
staining buffer to 1 x 106 cells.
Incubate 20 minutes on ice.
8. Wash cells in 2 ml staining
buffer and centrifuge (500 x g,
5 minutes).
9. Add secondary antibody
5 µg/ml in 100 µl staining
buffer. Incubate 30 minutes on
ice in the dark.
10.Wash cells in 2 ml staining
buffer and centrifuge (500 x g,
5 minutes).
11.Resuspend at 2 x 106/ml in cell
buffer and load on chip according
to the reagent kit guide.
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Conclusion
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Apoptosis is a critical mechanism
for organisms to maintain tissuehomoeostasis.
In this Application
Note we showed that the Agilent
2100 bioanalyzer is a versatile tool
to detect apoptotic cells. Protocols
and a list of recommended
reagents for detection of two
apoptotic markers are given.
Excellent reproducibility of
results from different chips for
both the Annexin V and the caspase-
3 assay is demonstrated. Data from the 2100 bioanalyzer
compares very well with that of a
conventional flow cytometer in
spite of a 5-fold lower cell consumption.
Data acquisition is done
automatically and data analysis is
achieved by an intuitive software
package which does not require
manual setting of instrument
related parameters.
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