Endothelial Cell Adhesion Assay Kit - Millipore

Endothelial Cell Adhesion
Assay Kit
Cat. No. ECM645
96 Tests
FOR RESEARCH USE ONLY
Not for use in diagnostic procedures
USA & Canada
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Introduction
Endothelial cells form the inner lining of blood vessels and serve as the cellular
interface between the circulating blood and the vessel wall. Endothelial cells are
involved in many aspects of vascular biology, including inflammation and
angiogenesis. The recruitment of leukocytes into inflammatory tissues is
regulated by the interaction between blood cells and endothelial cells that is
preceded by integrin-mediated cell adhesion. Endothelial-leukocyte cell adhesion
plays a major role in cellular communication and regulation, and is of fundamental
importance in the development and maintenance of tissues. Atherosclerosis, ulcer,
myocardial infarction, stroke, and other inflammatory processes are all related
to, and dependent on this endothelial-leukocyte adhesion process.
Expression of surface molecules on the vascular endothelium is altered at sites of
pathological inflammation. Endothelial Cell Adhesion Molecules (ECAMs) are
important mediators of leukocyte recruitment and adherence to the endothelium.
ECAMs such as E-selectin, VCAM-1, and ICAM-1 are upregulated during
inflammation, which initiates leukocyte adhesion to the endothelium, and
ultimately contributes to disease progression or tissue damage. Expression of
these ECAMs is mediated by proinflammatory cytokines such as Interleukin-1
beta and Tumor Necrosis Factor alpha, which propel the interaction of integrin
multimers on the leukocyte cell surface and the ECAMs on the endothelial cells.
The genes encoding ECAMs contain overlapping control mechanisms that allow
a single signaling pathway to upregulate several genes with the potential to
change the endothelial phenotype. Since ECAMs are regulated at the gene level
by transcription factors, proteasome inhibitors can modulate cytokine-induced
expression of ECAMs and subsequent leukocyte adhesion to the endothelium.
Controlling and manipulating the endothelial-leukoctye adhesion process will
create new avenues for drug therapies for vascular and inflammatory disease
states.
The CHEMICON ® Endothelial Cell Adhesion Assay kit (ECM645) allows the
researcher to test a variety of cell types that interact with activated or inactivated
endothelial cell layers. Human umbilical vein endothelial cells (HUVECs) from
ATCC ® (CRL-1730) or Cambrex © (CC-2519) are ideal for measuring
endothelial adhesion activity with the assay. The CHEMICON ® Endothelial
Cell Adhesion Assay kit includes a 96 well tissue culture treated fluorescence
microtiter plate and reagents that allow for large-scale screening and quantitative
comparison of multiple samples/cell line adhesion to an endothelial monolayer.
Once endothelial cells are seeded in the tissue culture plate, they can be treated
with cytokines, chemokines, transcription regulators, or selective adhesion
inhibitors. The protein synthesis inhibitor Cycloheximide and the RNA
synthesis inhibitor Actinomycin D are provided as controls for adhesion protein
expression studies. Treatment of endothelial cells, such as human umbilical vein
endothelial cells (HUVECs), with pro-inflammatory cytokines such as
1

Interleukin-1β or Tumor Necrosis Factor-α can induce expression of ECAMs, as
well as other adhesion molecules. The kit is supplied with both cytokines.
The viable cell fluorescent compound Calcein-AM ® is provided for labeling test
cells, such as leukocytes (granulocytes, monocytes, lymphocytes), fibroblasts,
neural stem cells etc., and quantifying their adhesion by comparing bound versus
unbound cells. The kit includes three Mouse anti-human monoclonal mAbs for
adhesion blocking studies of ICAM-1, VCAM, and E-selectin endothelial cell
surface markers. These may be used to verify changes in expression level of
adhesion molecules, or effects of integrins upon endothelium activation. This
simple and high-throughput kit format allows for the monitoring of the effects of
a variety of conditions and compounds on endothelial adhesion. CHEMICON ®
also offers a variety of antibodies to leukocyte adhesion molecules and surface
antigens. See CHEMICON ® ‘s website at www.chemicon.com for additional
information.
CHEMICON ® continues to provide numerous migration, invasion, and adhesion
products including:
• Endothelial Cell Characterization Kit (SCR023)
• In Vitro Angiogenesis Assay (ECM625)
• Fibrin In Vitro Angiogenesis Assay (ECM630)
• Blood Vessel Staining Kit (ECM590)
• Alpha Integrin-Mediated Cell Adhesion Colorimetric Array Kit (ECM530)
• Beta Integrin-Mediated Cell Adhesion Colorimetric Array Kit (ECM531)
• Alpha/Beta Integrin-Mediated Cell Adhesion Colorimetric Array Combo Kit
(ECM532)
• Alpha Integrin-Mediated Cell Adhesion Fluorimetric Array (ECM533)
• Beta Integrin-Mediated Cell Adhesion Fluorimetric Array (ECM534)
• Alpha/Beta Integrin-Mediated Cell Adhesion Array Combo Fluorimetric Kit
(ECM535)
• ECM Cell Adhesion Colorimetric Array Kit (ECM540)
• ECM Cell Adhesion Fluorimetric Array Kit (ECM545)
• QCM 8µm 96-well Chemotaxis Cell Migration Assay (ECM510)
• QCM 8µm 24-well Chemotaxis Cell Migration Colorimetric Assay
(ECM508)
• QCM 8µm 24-well Chemotaxis Cell Migration Fluorimetric Assay
(ECM509)
• QCM 5µm 96-well Chemotaxis Cell Migration Assay (ECM512)
• QCM 3µm 96-well Chemotaxis Cell Migration Assay (ECM515)
• QCM 96-well ECMatrix TM Cell Invasion Assay (ECM555)
• QCM 96-well Collagen-based Cell Invasion Assay (ECM 556)
2

Test Principle
The CHEMICON ® Endothelial Cell Adhesion Assay utilizes a homogenous
fluorescence detection format, allowing for large-scale screening and
quantitative comparison of multiple samples with the flexibility for testing a
variety of conditions. First, endothelial cells are seeded in the 96 well plate where
they grow to confluency. Prior to activation, the endothelium can be treated with
the protein synthesis inhibitor, (Cycloheximide) or the RNA synthesis inhibitor
(Actinomycin D) to control endothelial adhesion protein expression.
Subsequently, the endothelium is activated with one or two cytokines (TNF alpha
and Interleukin-1 beta) provided. Upon activation, endothelial cells can be used
for adhesion blocking studies with functional blocking mAbs to endothelial cell
adhesion markers (e.g. E-selectin, ICAM-1, VCAM). Next, test cells are labeled
with the Calcein AM ® green fluorescence viability cell marker and added to the
endothelium on the plate. After a brief incubation, unbound cells are washed away
while bound cells remain attached to the endothelium. Finally, the plate is read on
a fluorescence plate reader. Relative test cell adhesion is directly related to the
fluorescence of the wells. See the diagram on the next page.
Application
The CHEMICON ® Endothelial Cell Adhesion Assay Kit is a versatile tool for
assessing and quantifying cell to cell interaction of human endothelial cells with
leukocytes, or other cell types, under a variety of conditions. The kit is ideal for
the assessment of specific cell surface integrins and surface receptors on human
endothelial cells, monitoring in vitro cell differentiation, assessing endothelial
adhesion blocking, or screening potential cell adhesion promoters/inhibitors. A
variety of leukocytes or leukocyte-like cell lines can be tested in response to
manipulation of the endothelial cell behavior. Each CHEMICON ® Endothelial
Cell Adhesion Assay Kit contains sufficient reagents for the evaluation of 96
samples.
The CHEMICON ® Endothelial Cell Adhesion Assay Kit is intended for research
use only, not for diagnostic or therapeutic applications.
3

Human Endothelial
cells are seeded in a
black fluorescence tissue
culture treated plate.
Endothelial cell adhesion
molecules express on
the HUVEC surface.
Blocking antibodies
against ECAMs or
integrins can be added.
Endothelial Adhesion Assay(s) Procedure
The cells are cultured 48
to 72 hours or until they
are confluent.
4
The cells are treated with
a pro-inflammatory
cytokine such as TNFα
or IL1-β. The plate is
incubated for 2-6 hours.
TNFα
TNFα
TNFα
TNFα
TNFα TNFα
Calcein-AM labeled cells, such as
leukocytes, are added to the
wells. The plate is incubated
briefly to allow for cell binding.
Non-specific cells are washed
away. The plate is read at
485nm/530nm in a
fluorescent plate reader.
Fluorescence is a direct
correlation of cell adhesion.
Blocking antibodies inhibit or
reduce the leukocyte adhesion to
activated endothelial cells
For Research Use Only. Not for use in diagnostic procedures.
TNFα
TNFα
TNFα
TNFα
TNFα
Plate
Reader
Fluorescence

Kit Materials
1. 96-Well Endothelial Cell Tissue Culture Fluorescence Plate: (Part No.:
2004174) One 96-well tissue culture-treated black fluorescence plate with
lid.
2. Cycloheximide: (Part No. 2004165) One vial – 100 µL of solution.
3. Actinomycin D: (Part No. 2004166) One vial – 50 µg of crystals.
4. Tumor Necrosis Factor alpha: (Part No. 2004167) (Chemicon Cat. No.
GF023) One vial – 20 µL of solution.
5. Interleukin-1 beta: (Part No. 2004168) (Chemicon Cat. No. IL038) One
vial – 12 µL of solution.
6. 1X Assay Buffer: (Part No. 2004320) One bottle - 100 mL of solution.
7. Calcein AM Dye ® : (Part No. 2004169) One vial – 50 µL of solution, ready
to use.
8. Mouse anti-Human ICAM-1 Monoclonal Antibody: (Part No. 2004181)
(Chemicon Cat. No. MAB1379) One vial - 30 µL of solution.
9. Mouse anti-Human VCAM Monoclonal Antibody: (Part No. 2004182)
(Chemicon Cat. No. MAB2144) One vial - 30 µg in solution.
10. Mouse anti-Human E-selectin Monoclonal Antibody: (Part No. 2004183)
One vial – 37.5 µL of solution.
11. Mouse Negative Control IgG: (Chemicon Cat. No. PP54-100UG) One vial
- 100 µL of solution.
Materials Not Supplied
• Endothelial Cells such as HUVEC.
• Endothelial Cell Basal Medium (EBM) without FBS or FCS: Tissue culture
basal medium appropriate for subject endothelial cells or test cells.
• Tissue culture growth medium appropriate for subject endothelial cells or
test cells, such as Endothelial Cell Growth Medium (EGM), DMEM,
EMEM, or FBM (Fibroblast Basal Media) containing 10% FBS.
• Harvesting Buffer: EDTA or trypsin cell detachment buffer. Suggested
formulations include a) 2 mM EDTA/PBS, b) 0.05% trypsin in Hanks
Balanced Salt Solution (HBSS) containing 25 mM HEPES, or other cell
detachment formulations as optimized by individual investigators.
5

Note: Trypsin cell detachment buffer may be required for difficult cell lines.
Allow sufficient time for cell receptor recovery.
• Quenching Medium: Serum-free medium, such as EGM, DMEM, EMEM,
or FBM, containing 5% BSA
Note: Quenching Medium must contain divalent cations (Mg 2+ , Ca 2+ )
sufficient for quenching EDTA in the harvesting buffer
• Sterile PBS or Hank’s Balanced Salt Solution (HBSS) for washing cells
• Sterile pipettes and pipette tips sufficient for aliquoting cells
• Sterile cell culture hood
• Low speed centrifuge and tubes for cell harvesting
• CO2 incubator appropriate for subject cells
• Hemacytometer and microscope for counting cells
• Trypan blue or equivalent viability stain
• DMSO
• Multi-channel and/or repeating pipettes
• Deionized water
• Conical and microfuge tubes
• Fluorescence plate reader with 485 nm and 530 nm filters
Precautions
• No data is available on the biological toxicity of Calcein AM ® dye. This
chemical is membrane permeable and, as such, should be treated as a
potential mutagen, which may cause cancer and heritable genetic damage.
Handle with caution. DMSO stock solution should be handled with special
caution as DMSO can facilitate the entry of organic molecules into tissues.
• Cycloheximide is highly toxic and should be handled with care. Use
protective wear when handling. This chemical is membrane permeable and,
as such, should be treated as a potential mutagen, which may cause cancer
and heritable genetic damage.
• Actinomycin D is highly toxic, carcinogenic, teratogenic and should be
handled with care. Use protective wear when handling. This chemical is
membrane permeable and, as such, should be treated as a potential mutagen,
which may cause cancer and heritable genetic damage. DMSO stock
6

solution should be handled with special caution as DMSO can facilitate the
entry of organic molecules into tissues.
Storage
The Assay Buffer and the Endothelial Cell Culture Plate can be stored at 2° to 8°C
up to their expiration dates. Store the plate in its foil pouch. If not using the entire
plate, seal any used wells with a plate sealer to protect the unused wells. Ensure
that the desiccant remains in the pouch, and that the pouch is securely closed.
Store the remaining kit components at -20°C. Prepare undiluted aliquots for the
Cycloheximide, TNF alpha, and IL-1 beta. Avoid repeated freeze-thaw cycles.
Note: Actinomycin D adsorbs to plastic and glass upon standing in solution;
however, frozen aliquots of the stock vial are expected to be stable for at least
30 days at -20°C.
Preparation of Reagents
Important Note: During shipment, small volumes of product will occasionally
become entrapped in the seal of the product vial. For products with volumes of
200 µL or less, we recommend briefly centrifuging the vial in a tabletop
centrifuge to dislodge any liquid in the container’s cap.
Reagent Product Description Preparation
1 Cycloheximide: Blocks translation of
messenger RNA on cytosolic
80S ribosomes, but does not
inhibit organelle protein
synthesis.
2 Actinomycin D: Inhibits cell proliferation by
forming a stable complex
with dsDNA, thus inhibiting
DNA-primed RNA synthesis.
7
Cycloheximide is
provided at 10
mg/mL. Prepare a
solution of
Cycloheximide to 1-
20 µg/mL in Assay
Buffer or media as
needed.
Actinomycin D is
provided as 50 µg
crystals.
Reconstitute the vial
with 50 µL of
DMSO. Vortex
thoroughly. Prepare
a solution of
Actinomycin D to 1-
10 µg/mL in Assay
Buffer or media as
needed.

3 Tumor Necrosis
Factor alpha
(TNFα):
4 Interleukin-1
beta (IL-1β):
5 Mouse anti-
Human ICAM-1
Monoclonal
Antibody:
TNFα is a pleiotropic
inflammatory cytokine. The
protein acts as a key mediary
in the local inflammatory
immune response. TNFα
initiates a cascade of
cytokines, ECAMs, and
increases vascular
permeability, thereby
recruiting leukocytes to a site
of infection.
IL-1β is a pleotropic
cytokine, secreted primarily
by monocytes and
macrophages, that mediates
the pathophysiology of
various acute and chronic
inflammatory conditions. IL-
1β’s pro-inflammatory effects
are modulated by increased
endothelial vascular
permeability, accompanied
by ECAM and cytokine
expression.
Mouse IgG 1 mAb recognizes
the extracellular D1 domain
of ICAM-1. Reacts with
ICAM-1 antigen found on
lyphocytes, monocytes,
granulacytes, fibroblasts, and
endothelial cells. Blocks
ICAM-1 mediated adhesion
to LFA-1.
8
TNFα is provided at
0.1 mg/mL. Prepare
a solution of TNFα
to working
concentration range
of 10-1000 ng/mL in
Assay Buffer or
media as needed, just
prior to use.
IL-1 beta is provided
at 0.1 mg/mL.
Prepare a solution of
Interleukin-1 beta to
a working
concentration range
of 10-1000 ng/mL in
Assay Buffer or
media as needed, just
prior to use.
Ms x ICAM-1 is
provided at 1 mg/mL.
Dilute in Assay
Buffer or media as
needed. Suggested
working
concentration is 10-
50 µg/mL.
Note: The end user
must determine the
optimal
concentration and
working dilutions for
their particular
application.

6 Mouse anti-
Human VCAM
Monoclonal
Antibody:
7 Mouse anti-
Human
E-selectin
Monoclonal
Antibody:
Mouse IgG 1 mAb recognizes
IL-1β activated endothelial
cells and inhibits cell
adhesion.
Mouse IgG 1 mAb recognizes
E-selectin expressed on
cytokine activated HUVECs
and inhibits adhesion of
neutrophils, eosinophils, and
skin-homing T-cells. This
mAb has been shown to block
U937, HL-60, THP-1, as well
as HUVECs. This mAb
binds specifically to Eselectin
after being screened
against COS cells transfected
with cDNAs for E-selectin, Pselectin,
L-selectin, PECAM-
1, ICAM-1, and VCAM-1.
9
Ms x VCAM is
provided at 1 mg/mL.
Dilute in Assay
Buffer or media as
needed. Suggested
working
concentration is 10-
50 µg/mL.
Note: The end user
must determine the
optimal
concentration and
working dilutions for
their particular
application.
Ms x E-selectin is
provided at 1 mg/mL.
Dilute in Assay
Buffer or media as
needed. Suggested
working
concentration is 20-
50 µg/mL.
Note: The end user
must determine the
optimal
concentration and
working dilutions for
their particular
application.

8 Mouse Negative
Control IgG:
Mouse IgG whole molecule
can be used as a negative
control on the endothelium to
determine background
affinity or non-specific
binding of test cells.
Preparation of Endothelial Cells
Perform the following steps in a sterile hood
10
Ms IgG is provided
at 1 mg/mL. Dilute
in Assay Buffer or
media as needed.
Note: The end user
must determine the
optimal
concentration and
working dilutions for
their particular use.
Prepare Endothelial cell line for investigation as desired. The following
procedure is a suggestion for preparing endothelial cells used in cell layer
formation. Human umbilical vein endothelial cells (HUVECs) from ATCC ®
(CRL-1730) or Cambrex © (CC-2519) were used for generating endothelial
adhesion activity data for this assay.
1. Use cells that are at least 80-100% confluent.
2. Visually inspect cells before harvest, taking note of relative cell numbers
and morphology.
3. Wash cells 2 times with sterile PBS or HBSS.
4. Add 5 mL Harvesting Buffer (see Materials Not Supplied) per 100 mm dish
and incubate at 37˚C for 5-15 minutes.
5. Add 10-20 mL of Quenching Medium or Endothelial Cell Growth Medium
(see Materials Not Supplied) to inactivate trypsin/EDTA from Harvesting
Buffer and gently pipet the cells off the dish.
6. Centrifuge cells gently to pellet (400 x g, 5-10 minutes).
7. Carefully remove media from pellet.
8. Gently resuspend the pellet in 1-5 mL of Quenching Medium or Endothelial
Cell Growth Medium, depending upon the size of the pellet. Optimum cell
density may be determined by titration of the cells. Often it is best to harvest a
greater number of cells than is needed.
9. Count cells and bring to a volume that provides a concentration of
1.0 –2.0 x 10 6 cells/mL with growth or basal media (EGM or EBM).
10. OPTIONAL: If pretreatment of Endothelial Cells is desired, add
compound(s) (cytokines, pharmacological agents, etc.) at this time.

Preparation of Calcein AM ® Labeled Test Cells
Perform the following steps in a sterile hood
Prepare cell line for investigation as desired. The following procedure is a
suggestion for preparing leukocytic cell lines used in cell adhesion.
Note: Use Calcein AM ® labeled cells within 4 –12 hours.
1. Use cells that are at least 80-100% confluent.
2. Visually inspect cells before harvesting, taking note of relative cell numbers
and morphology.
3. Dissociate adherent or non-adherent cell lines to be tested into separate,
single cell suspensions.
4. Dilute the leukocyte cell suspension(s) to 5 mL.
5. Add 12.5 µL of Calcein AM ® to the cell suspension(s) for a 2.5 µM final
concentration. Mix gently by inversion.
6. Incubate the cells in a 37˚C CO2 incubator for 30 minutes.
7. Centrifuge cells gently to pellet (400 xg, 5-10 minutes).
8. Carefully remove the media from the cell suspension(s).
Note: The cell pellet(s) should have a bright yellow appearance.
9. Wash each labeled cell line with 5-10 mL of PBS or HBSS. Pipette to
resuspend and wash the cells thoroughly.
10. Centrifuge cells gently to pellet (400 xg, 5-10 minutes). Carefully remove
and discard the wash.
11. Repeat wash 2-3 times until all residual Calcein AM ® is removed.
12. After the last wash, gently resuspend the cells in 1-5 mL of Assay Buffer,
depending upon the size of the cell pellet.
13. Count the cells and bring to a volume that provides a concentration of
0.5 –2.0 x 10 6 cells/mL with Assay Buffer or media.
Note: Resuspend the cells if they stand too long and settle prior to using
them.
Assay Instructions
Perform the following steps in a sterile hood
Note: Prepare cell lines and reagents for investigation as desired. The
following procedure is a suggestion for performing leukocyte-endothelial cell
adhesion. This procedure is intended as a guide. The user is encouraged to
define their experimental parameters when evaluating endothelial adhesion.
11

1. Rehydrate the desired number of plate wells with 100 µL of EGM per
well and incubate the plate at room temperature until the cell
suspensions are ready.
2. Spin the final single endothelial cell suspension down one more time
and gently resuspend in EGM. See Cell Harvesting.
3. Prepare endothelial cells to desired concentration in EGM. A common
starting range is 0.5 to 5.0 x 10 5 cells/mL.
4. Add 100 µL of the endothelial cell suspension to each plate well to be
tested. It is recommended that you seed 5,000 to 50,000 endothelial
cells per well and prepare enough wells to assay each sample in
duplicate or triplicate.
5. Incubate the plate for 48-72 hrs at 37°C in a CO 2 incubator. During the
incubation, monitor the cell growth microscopically to ensure cell
viability, morphology and uniformity.
6. Change the media every 24-48 hours.
Optional: Perform cell starvation if desired. Incubate endothelial cells
with basal media (EBM) with 1-2% serum for 4-24 hours prior to
activation.
7. After incubation, gently discard or aspirate the media from the wells.
Note: Do not allow wells to dry.
8. Add 100 µL of Cycloheximide or Actinomycin D solutions to control
wells.
9. Add 100 µL of media to the non-treated wells.
10. Cover and incubate the plate 30 minutes at 37°C in a CO 2 incubator.
11. Activate the endothelial cells by adding 10 µL of Tumor Necrosis
Factor alpha or Interleukin-1 beta to the appropriate test wells. This
dilutes the TNFalpha and IL-1beta to a final concentration range of ~1-
100 ng/mL.
12. Cover and incubate the plate at 37°C in a CO 2 incubator for 2-6 hours.
13. Carefully remove 75-100% (~75 µL) of the solution from the wells and
discard.
Note: Do not allow wells to dry.
14. Gently wash each well 1-2 times with 200 µL per well of Assay Buffer.
Remove each 200 µL wash and discard. Try to leave about 20% (~50
µL) of Assay Buffer per well when aspirating/removing the last wash
solution so the cells remain hydrated at all times.
Note: If performing endothelial adhesion blocking studies with a
blocking mAb, go to step 15. If not performing endothelial adhesion
blocking with a blocking mAb, go to step 17.
15. Add adhesion-blocking mAb to the appropriate final concentration per
well.
16. Cover and incubate the plate at 37°C in a CO 2 incubator for an
appropriate time (~30-60 minutes).
12

Optional: The user may co-incubate the blocking mAb with the test
cell line.
17. Prepare test cell suspension as desired. Add 100 µL of test cell
suspension (~50,000 to 200,000) to each well. See Preparation of
Calcein AM ® Labeled Cells.
18. Cover and incubate the plate 30 minutes at 37°C in a CO 2 incubator.
19. After the incubation period, verify microscopically that the test cells
have settled onto the endothelium.
20. Carefully aspirate 75% of the solution from the wells and discard.
21. In order to remove test cells with non-specific binding, gently wash
each well 2-3 times with 200 µL per well of Assay Buffer. Remove
each 200 µL wash and discard. Try to leave about 50-100 µL of Assay
Buffer per well when aspirating/removing the wash solution so majority
of the endothelial/test cell adhesion is maintained, and only nonspecifically
bound test cells are removed.
Note: Use caution when washing as forceful pipetting can dislodge
cells and affect assay results.
22. After washing, leave 100 µL of Assay Buffer in each well.
23. Read the plate with a fluorescence plate reader using 485/530 nm
excitation/emission filter sets.
13

Row
A
B
C
D
E
F
G
H
Note: No Txt = No treatment.
Fig. 1 Suggested Experimental Adhesion Plate Layout: Suggested
experimental design for testing four different cell lines utilizing all of the kit
components. This plate layout is intended as a guide only. The end user must
determine their optimal working conditions and protocols.
Calculation of Results
Suggested Adhesion Plate Layout
Well Number
Column 1 2 3 4 5 6 7 8 9 10 11 12
Test Cell →
-----------------
Condition
↓
Cycloheximide
→
Actinomysin
D →
Ms x ICAM-1
→
Ms x VCAM
→
Ms x Eselectin
→
Ms IgG
→
Negative
→
Negative
→
Test
Cell
#1
↓
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
Test
Cell
#1
↓
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
Test
Cell
#2
↓
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
Test
Cell
#2
↓
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
Test
Cell
#3
↓
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
Optimal assay timing and performance may vary for different cell lines but
generally can be obtained using subconfluent cell cultures. Subconfluent cultures
can be achieved by splitting cells 1 to 2 days prior to performing the assay.
Results of the Endothelial Cell Adhesion Assay may be illustrated graphically by
the use of a "bar" chart. A typical endothelial cell adhesion experiment will
compare the activated endothelium against inactivated cells. Results from
negative control wells are typically used as fluorescence background for
interpretation of data. A small amount of background fluorescence, or "noise,"
is expected from the negative wells.
14
Test
Cell
#3
↓
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
Test
Cell
#4
↓
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
Test
Cell
#4
↓
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
TNF
or
IL1β
Test
Cell
#1
↓
No
Txt
No
Txt
No
Txt
No
Txt
No
Txt
No
Txt
No
Txt
No
Txt
Test
Cell
#2
↓
No
Txt
No
Txt
No
Txt
No
Txt
No
Txt
No
Txt
No
Txt
No
Txt
Test
Cell
#3
↓
No
Txt
No
Txt
No
Txt
No
Txt
No
Txt
No
Txt
No
Txt
No
Txt
Test
Cell
#4
↓
No
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No
Txt
No
Txt
No
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No
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No
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No
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No
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The following charts illustrate typical results for the various cell lines
tested. This data should be used for reference only and not be used to
interpret actual assay results. The end user must determine optimization
and experimental conditions.
Relative Fluorescence Units (RFUs)
140000
120000
100000
80000
60000
40000
20000
0
Adhesion of HL60 and THP-1 Cells to HUVECs
(+)
Cycloheximide
(+) Cytokine
(+)
Cycloheximide
(-) Cytokine
(-)
Cycloheximide
(+) Cytokine
Adhesion Assay Conditions
HL60 IL-1beta
Treatment
HL60 TNFalpha
Treatment
THP-1 IL-1beta
Treatment
THP-1 TNFalpha
Treatment
(-)
Cycloheximide
(-) Cytokine
Fig. 2 Leukocyte-Endothelial Adhesion Inhibition. Human Vascular Endothelial
cells (HUVECs) were cultured until confluency and then treated with or without
Cycloheximide. The cells were then activated with TNF alpha or IL-1 beta for 4
hours at 37°C. Next, Calcein AM ® labeled HL60 and THP-1 cells were incubated for
30 minutes at 37°C. Cycloheximide blocked >90% HL60 and THP-1 cell adhesion
to the endothelium compared to treated and non-treated HUVECs. Relative
Fluorescence Units were determined using a Perkin Elmer Victor 2 1420 Multilabel
Counter.
References
1. Béchard, D. et al. (2001). J. Immunology. 167: 3099-3106.
2. Bevilacqua, M. et al. (1985). J. Clin. Invest. 76: 2003-2011.
3. Bevilacqua, M. et al. (1986). Proc. Natl. Acad. Sci. 83: 4533-4537.
4. Carlos, T. and Harlan J. (1994). Blood. 84(7): 2068-2101.
15

5. Dagia, N. and Goetz, D. (2003). Am. J. Physiol. Cell Physiol. 285: C813-
C822.
6. Etzioni A., Doerschuk C., and Harlan J. (1999). Blood. 94(10): 3281-3288.
7. Harlan, J. and Winn, R. (2002) Crit. Care Med. 30: S214-S219.
8. Katagiri, K. et al. (1996). Blood. 87(10): 4276-4285.
9. Luscinskas F. and Gimbrone M. (1996) Annu. Rev. Med. 47: 413-421.
10. Nozawa, F. et al. (2000). Pancreas. 21(4): 392-398.
11. Panés J., Perry M., and Granger DN. (1999) British J. Pharm. 126: 537-
550.
12. Pober J. et al. (1986). J. Immunol. 136: 1680-1687.
13. Read, M. et al. (1995). Immunity. 2: 493-506.
14. Shen, J. et al. (1997). J. Virology. 71(12): 9323-9332.
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CHEMICON ® literature when used in accordance with their instructions.
THERE ARE NO WARRANTIES, WHICH EXTEND BEYOND THIS
EXPRESSED WARRANTY AND CHEMICON ® DISCLAIMS ANY IMPLIED
WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS
FOR PARTICULAR PURPOSE. CHEMICON ® ’s sole obligation and
purchaser’s exclusive remedy for breach of this warranty shall be, at the option
of CHEMICON ® , to repair or replace the products. In no event shall
CHEMICON ® be liable for any proximate, incidental or consequential damages
in connection with the products.
©2006: CHEMICON ® International, Inc. - By CHEMICON ® International, Inc.
All rights reserved. No part of these works may be reproduced in any form
without permissions in writing.
16

Cat No. ECM645
March 2006
Revision A: 4001980