Epithelial Cells and Pathogens - The Odyssey® System
Brings Light into the Darkness
Manuel Amieva and Roger Vogelmann
Stanford University
Principle: The barrier function of tight junctions
in mammalian epithelial cells can be measured
by plating them in Transwell®-filters on polycarbonate membranes (Costar Corp., Cambridge,
MA, USA). In a confluent monolayer of epithelial
cells, media in the apical compartment is separated from media in the basal compartment. A tracer
is added to the apical media and media from the
basal compartment is analyzed for tracer material
indicating leakage of the barrier of the cell monolayer. Fluorochrome labeled dextran molecules in
various sizes (3 - 40 kD), which can be detected in
the 700 or 800 nm channel of the Odyssey® Infrared Imaging System, can be used as a tracer.
0h
1h
3h
6h
9h
12h
bl
an
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TIGHT JUNCTION BARRIER
FUNCTION IN EPITHELIAL CELLS
apical
basal
apical
basal
Figure 1. Example for tight junction formation at time points
indicated after calcium-switch in confluent MDCK cell monolayer. 50 ml of 10 kD dextran Alexa Fluor 647 in DMEM media/well; scanned with Odyssey Infrared Imaging System at
700 nm wavelength. Notice that the tracer amount in the
basal compartment is decreasing during barrier formation.
Experiment: Epithelial MDCK cells are plated
in low calcium media (5 µM Ca2+) on 12 mm Transwell®-filters on 0.4 µm polycarbonate membranes
at confluent density (~ 1 x 106 cells/well). Filters
are pre-coated with collagen to increase the adhesiveness of the cells. After 30 min., cells are transferred into regular DMEM media (1.8 mM Ca2+)
to initiate cell-cell contact formation. At various
time points, media is replaced as follows:
■ 200 µl regular DMEM media with 100 µg/ml
10 kD dextran Alexa Fluor® 647 (Molecular
Probes, Eugene, OR, USA)
Dextran stock solution: 2 mg in 1 ml DMEM without FBS
● Dilute stock solution 1:20 in media for apical compartment
●
■ 400 µl regular DMEM media in basal compartment
Incubate for 30 minutes at 37 ºC in cell incubator. Collect 50 µl media of apical and basal compartment and transfer to 96-well plate. Analyze
data with Odyssey® Infrared Imaging System at
appropriate wavelength and analyze pixel intensity of wells (Figure 1). The amount of tracer in
apical and basal compartment can be determined
for each well by analyzing a dilution series of input material in apical compartment (Figure 2).
Published May, 2004 by LI-COR Biosciences.
Figure 2. Dilution series of 10 kD dextran Alexa Fluor® 647
in DMEM media; 50 ml/well; scanned with Odyssey Infrared
Imaging System at 700 nm wavelength. Integrated intensity
analyzed for each well (values are background corrected using integrated intensity for DMEM only); samples in triplicate. The tracer amount in basal compartment can be calculated using equation of the linear trend line.
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Manuel Amieva and Roger Vogelman
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Manuel Amieva
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Experiment:
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In this particular experiment, antibodies from
two different species against CagA were available.
We stained the membranes with both antibodies to
increase the sensitivity of the screen by identifying
positive clones that were stained in both channels.
We used two picture printouts of the scan to identify
positive clones on the blood agar plate. One printout
IB: CagA, pc
merge
wildtype CagA
IB: CagA, mc
●
all colonies on plate
merge
Coomassie
CagAEPISA clone 1
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CagAEPISA clone 2
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We generated CagA knockout clones of H. pylori
by homologous recombination with an antibiotic
resistant gene cassette.
A mutant of the CagA protein (CagAEPISA) was reintroduced into the bacterial genome by natural
transformation and homologous recombination.
To screen for positives clones, nitrocellulose membranes were cut into circles that fit 10 cm Petri
dishes.
The membrane was placed on top of the bacterial colonies growing on blood agar. (TIP: To avoid
bubbles when you place the membrane on agar
CagAEPISA clone 1
●
wildtype CagA
Principle: Scanning of bacterial clones with the
Odyssey Infrared Imaging System helps to identify
clones that are positive for a certain protein/DNA.
plate, wait until membrane is completely wet.)
Lift membrane from plate and let it air dry, followed by UV cross-linking.
Put Petri dish back into incubator so that bacterial colonies can grow back.
Permeabilize bacteria on membrane by incubating in PBS + 0.1% Triton X-100 for 5 minutes at
room temperature (RT).
Wash in PBS and block with 5% skim milk in
PBS for 1h at RT.
Follow standard western blotting protocol for Odyssey Infrared Imaging System to incubate with
primary and secondary antibody.
CagAEPISA clone 2
EXPRESSION CLONING OF
BACTERIAL CLONES
IB CagA
Figure 3. Expression cloning of H. pylori CagAEPISA strains. Membranes stained with antibodies against
CagA (mc - mouse monoclonal [green]; pc – rabbit polyclonal [red]). Colonies on plate are pseudo-colored
in white. Two clones picked and analyzed with SDS-gel electrophoresis – total protein detected with Coomassie blue staining; immunoblot stained with anti-CagA mc antibody (green).
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Epithelial Cells and Pathogens - The Odyssey System Brings Light into the Darkness
was adjusted so that only positive clones in both channels would be visible. The other printout showed all
colonies by increasing the electronic sensitivity of the
scan. With these printouts, we were able to identify
and pick the positive clones on the plate. If only one
antibody against a protein of interest is available, the
second channel can be used to stain with an antibody
that detects all bacteria to help with the orientation
on the plate.
Often antibodies against proteins of interest are
not available. The experiment can be modified using fluorochrome labeled oligonucleotides following a
southern-blot like protocol.
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Wash gently 3 x with PBS.
Analyze 96-well plate with Odyssey Infrared
Imaging System at 700 nm wavelength.
In this particular experiment, we analyzed
two H. pylori strains, one non-adherent strain
G27 and one adherent strain G27-MA (Figure 4).
We also treated some wells (columns 4-6 and 1012) with PBS for 30 min. at 37 ºC/ 5% CO2 before
adding bacteria to loosen cell-cell contacts and
consequently to increase the attachment area. As
shown in Figure 4, some of the cell monolayers
were disrupted by the washing steps, but those
wells were not taken into account for the data
analysis (see colored boxes).
ADHESION ASSAY TO ASSESS
BACTERIAL ATTACHMENT TO
EPITHELIAL CELLS
Manuel Amieva
A)
Principle: The Odyssey Infrared Imaging System can be used to determine the amount of bacteria attached to epithelial cells.
Experiment: MDCK cells are grown as a confluent monolayer in 96-well plates. Therefore,
~ 3 x 105 cells/well were plated one day before the
experiment. Different amount of H. pylori were
incubated with cells. Therefore,
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Make dilution series of bacteria in growth media for cells.
Incubate bacteria with cells for 1 hr. in cell incubator at 37 ºC / 5% CO2.
Remove media and wash carefully 3 x with
PBS at RT.
Fix cells in 2% paraformaldehyde for 15 min.
at RT.
Wash gently 3 x with PBS.
Block with PBS + 3% BSA for 10 min. at RT.
Stain with primary antibody directed against
outer membrane proteins of H. pylori for 1 hr
at RT (Important: for intracellular proteins
permeabilize bacteria first with 0.1% Triton
X-100 or 1% Saponin).
Wash gently 3 x with PBS + 3% BSA.
Add secondary antibody Alexa Fluor 680 for
30 min. at RT (1:1000 in PBS + 3% BSA).
Figure 4. H. pylori attachment to MDCK cells. G27 – non-adherent strain; G27-MA – adherent strain; MOI – multiplicity
of infection. ‘PBS’ indicates MDCK cells pre-treated with PBS
for 30 min. before bacterial attachment to increase attachment area.
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Manuel Amieva and Roger Vogelman
B)
Principal: The number of attached bacteria can
be estimated from the Integrated Intensity level
of the scan data.
Experiment:
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Take half of a bacterial solution and determine
number of bacteria using standard protocol for
‘colony forming units’ (in triplicate).
Take second half of bacterial solution for adhesion assay following protocol A).
Protocol A) is modified in a way that we use
AGS cells, which have a very high affinity for
binding H. pylori.
Determine Integrated Intensity for attached
bacteria using the Odyssey Infrared Imaging
System (in triplicate).
Blot CFU value against Integrated Intensity
level in xy-graph (Figure 5).
The number of attached bacteria (CFU) can be
estimated based on the Integrated Intensity using the equation of the linear trend line (Figure 5;
note that the Integrated Intensity is linear to the
log scale of CFU).
Figure 5. Attachment of H. pylori to AGS cells. Integrated Intensity and CFU (colony forming units) measured
in triplicate. G27-MA and CA-1 are two separate H. pylori strains.
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LI-COR, inc. Alexa Fluor is a registered trademark of Molecular Probes. Transwell is a registered trademark of Corning-Costar Corporation. The
Odyssey Infrared Imager and IRDye infrared dyes are covered by U.S. patents (6,495,812, 6,593,148 and 6,027,708) and patents pending.
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