Detection of antibody-stained cell
surface and intracellular protein targets
with the Agilent 2100 bioanalyzer
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This Application Note describes how the Agilent 2100 bioanalyzer and
the Cell Fluorescence LabChip® kit can be used to detect cell surface
and intracellular protein targets by antibody staining. CD3 staining on
Jurkat cells as well as CD4 staining on CCRF-CEM cells and intracellular
glucocorticoid receptor (GR) staining of H4 rat hepatocytes were
performed. Histogram quality and the percentage of stained cells counted
with the microfluidic system are in good agreement with data
obtained with a conventional flow cytometer. Detailed protocols and
reagent recommendations for staining cell surface and intracellular
protein targets are given. The high reproducibility of the chip results,
low cell consumption and ease-of-use are advantages of the compact
bioanalyzer system for monitoring cell surface and intracellular protein
target expression by antibody staining.
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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.
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Antibody staining
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Monitoring cellular protein
expression is a critical step for
quality control and characterization
of cell populations or assay
optimization and can be achieved
by staining the protein of interest
with a specific antibody. These
specific reagents can either be
directly labeled with a fluorescent
probe or they can be detected
with a secondary fluorescent reagent. An antibody is defined as
“an immunoglobulin capable of
specific combination with the antigen
that caused its production in a
susceptible animal” and usually
consists of a pair of two light and
two heavy amino acid chains.
They are produced in response to
the invasion of foreign molecules
in the body. Antibodies can be
divided into five subclasses based
on their heavy chain: IgG, IgM,
IgA, IgD and IgE. The most
commonly used antibody for specific
protein detection is IgG,
which can be cleaved by the protease
papain into three parts, two
F(ab) regions and one Fc, or into
two parts, one F(ab')2 and one Fc
by the proteolytic enzyme pepsin.
The F(ab) regions comprise the
“arms” of the antibody, which are
critical for antigen binding. The Fc
region comprises the “tail” of the
antibody and plays a role in
immune response.
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Cells carry antigens (lipids, proteins
or carbohydrates) which can
be detected with specific antibodies.
This can be achieved by treating
cells with specific antibodies
recognizing, for example, the protein
of interest. The detection antibody
may be directly conjugated
to a fluorescent probe, for example
allophycocyanine (APC), or a
fluorescently-labeled secondary
antibody may be used which
specifically binds to the Fc-part of
the primary antibody. The application
of antibody staining is not
restricted to the use of antigens
that are expressed on the cell surface.
Following fixation and permeabilization
of cells, intracellular
antigens can be easily detected by
antibody staining. Detection of proteins generally
depends on the availability of a
suitable antibody, many of which
are commercially available in a
fluorescently conjugated form. As
the Agilent 2100 bioanalyzer has
the highest sensitivity in the red,
with an excitation wavelength of
635 nm and a detection wavelength
of 685 nm, APC, or Cy5®
can be used as dyes. Counterstaining
the cells with the live cell dye
calcein allows the discrimination
between live and dead antibody
stained cells. In contrast, the measurement
of intracellular proteins
requires permeabilization and fixation
of cells prior to antibody
labeling. Therefore, staining with
Calcein is not advisable. In this
case cells can be counterstained
with the nucleic acid-specific-dye
SYTO16®.
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Staining of cell surface proteins
with antibodies may introduce
artifacts that should be eliminated
by special experimental procedures
and by carefully and critically
examining the results obtained.
On the one hand, crosslinking of
several receptor molecules by
bivalent antibodies may lead to
receptor internalization, where the
receptor becomes internalized
together with the bound antibodies
as described for endothelial
growth factor (EGF)-Receptor 1.
On the other hand, the extracellular
part of the receptor molecule
may be shed from the cell surface,
as described for CD442, 3. As these
effects require membrane alterations,
they are prevented if the
cells are fixed or kept continuously
on ice and in a buffer containing
low amounts of sodium azide.
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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 antibody-stained 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. 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 the intuitive
software package supplied with the
Cell Assay Extension with no
requirement to manually set instrument-
specific parameters.
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Results
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a) Extracellular CD3 staining of Jurkat
cells
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Jurkat cells, a cell line derived from
an acute human T-cell leukemia,
were harvested and washed. After
staining with calcein, the cells were
incubated with APC-labeled anti
hCD3-antibody. After washing and
resuspending the cells in cell buffer
the samples were loaded onto the
chip and measured in the Agilent
2100 bioanalyzer. Figure 1 shows
representative histograms of a mixture
of calcein-only and calcein and
antibody treated cells. The data
obtained with the 2100 bioanalyzer
are of comparable quality as data
generated with the same samples
on a conventional flow cytometer.
Figure 2 shows the reproducibility
of the measurement over several
chips and compares the data
obtained from the Agilent 2100 bioanalyzer
with flow cytometer measurements.
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b) Extracellular CD4 staining of
CCRF-CEM cells
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CCRF-CEM cells, a cell line
derived from an acute human lymphoblastic
leukemia, were harvested
and washed. After staining with
calcein, the cells were incubated
with APC-labeled anti hCD4-antibody.
After washing and resuspending
the cells in cell buffer the
samples were loaded onto the
chip and measured in the Agilent
2100 bioanalyzer. Figure 3 shows
representative histograms of a
mixture of calcein-only and calcein
and antibody treated cells.
These data are of comparable
quality as data generated with the
same samples on a conventional
flow cytometer. Figure 4 shows
the reproducibility of the measurement
over several chips and compares
the 2100 bioanalyzer data
with that obtained from the conventional
flow cytometer.
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c) Intracellular glucocorticoid receptor
(GR) staining of H4 hepatocytes
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For intracellular detection of GR
proteins H4 rat hepatocytes were
harvested and washed. The cells
were first stained with SYTO16®,
washed again and subsequently
treated with a permeabilizing/fixation
solution. They were then
stained with anti GR antibodies
and APC-conjugated secondary
antibody or APC-conjugated
secondary antibody alone. Mixtures
of both populations were
prepared at various ratios and
measured on the 2100 bioanalyzer
(figure 5) and on a conventional
flow cytometer. The 2100 bioanalyzer
displayed good reproducibility
and yielded data comparable to
the flow cytometer data (figure 6).
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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
L-glutamine
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a) Extracellular staining of CD3 or CD4
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Reagents
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• anti hCD3 APC antibody, anti
hCD4 APC antibody
• Calcein-AM (diluting the
original stock with DMSO to
yield a 500 µM solution)
• Staining buffer (PBS, 2% BSA,
0.05 % NaN3)
• Dye loading buffer (HBSS,
20 mM HEPES, 1 % BSA)
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Protocol
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1. Wash cells once in dye loading
buffer after harvesting.
2. Stain cells with calcein (up to
5 x 106/ml, 0.5 µM calcein,
15 minutes, 37 °C) in dye loading
buffer.
3. Wash once in staining buffer.
4. Stain cells (1 x 106) in a total
volume of 100 µl (80 µl staining
buffer and 20 µl antibody) for 25
minutes on ice in the dark. For
more cells (up to 5 x 106), adjust
amount of antibody but don't
increase total volume of 100 µl.
5. Wash twice with 1 ml staining
buffer.
6. Optional step if cells are to be
stored overnight and measured
later: Resuspend cells well at a cell
density of 1 x 106 cells /ml in
PBS. Make sure there are no cell
clumps in the suspension. Add 70 µl paraformaldehyde
(16 %-solution) while stirring.
Incubate for 10 minutes at
room temperature (RT) and
store sample at 4 °C in the
dark.
7. Resuspend cells in cell buffer
at 2 x 106 cells/ml and load
onto the chip.
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b) Intracellular GR staining of H4
hepatocytes
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Reagents
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• BD FACS Permeabilizing
Solution 2 (500 tests)
Alternatively, use Triton-X100
and Paraformaldehyde 16 % for
fixation and permeabilization.
• Anti rat GR antibody
• Phycoprobe AP Anti-Mouse IgG
• SYTO16
• Staining buffer (PBS, 2 % BSA,
0.05% NaN3)
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Protocol
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1. Harvest cells and adjust cell
density to 1 x 106 cells /ml in
PBS.
2. Add 1.5 µl SYTO16/ml and
incubate 10 minutes at 37 °C in
the dark.
3. Wash cells in 2 ml staining
buffer and centrifuge (500 x g,
5 minutes).
4. Resuspend cells in 500 µl
1x BD permeabilizing solution
(dilute in distilled water) and
incubate 10 minutes at RT.
5. Two alternative steps, if permeabilization and fixation
is done without BD permeabilizing
solution 2:
Resuspend in 1 ml HBSS.
Add 125 µl paraformaldehyde
(16 %-solution) and incubate 10
minutes on ice. Add 75 µl Triton (1 %-solution in
distilled water) and incubate 10
minutes on ice.
6. Wash cells in 2 ml staining
buffer and centrifuge (500 x g,
5 minutes).
7. Stain cells (1 x 106) with GR
antibody in a total volume of
100 µl. Add antibody to a final
concentration of 15 µg/ml (6 µl
ad 100 µl staining buffer) and
incubate for 30 minutes at RT.
For more cells (up to 5 x 106)
adjust amount of antibody but
do not in-crease total volume of
100 µl.
8. Wash cells in 2 ml staining
buffer and centrifuge (500 x g,
5 minutes).
9. Stain cells (1 x 106) with
secondary antibody (Phycoprobe
AP anti-mouse IgG) in a
total volume of 100 µl. Add
secondary antibody to a final
concentration of 15 µg/ml
(1.5 µl and 100 µl staining
buffer) and incubate for 30
minutes at RT. For more cells
(up to 5 x 106) adjust amount
of antibody but do not increase
total volume of 100 µl.
10.Wash cells two times in 2 ml
staining buffer and centrifuge
(500 x g, 5 minutes).
11. Resuspend at 2 x 106/ml in cell
buffer and load onto the chip.
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Conclusion
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Monitoring cellular expression of
protein targets is a critical step for
quality control, characterization of
cell populations or assay optimization.
In this Application Note we
demonstrated that the Agilent
2100 bioanalyzer is a versatile tool
to detect expression levels of surface
as well as intracellular protein
targets. Protocols and a list of
recommended reagents for detection
of two cell surface proteins
and one intracellular protein are
given. Excellent reproducibility of results from different chips is
demonstrated. The Agilent 2100
bioanalyzer shows a high sensitivity,
as demonstrated by the detection
of small subpopulations
expressing the target proteins
with only 5% of all cells. Data
from the 2100 bioanalyzer compares
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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