In Vitro Vascular Permeability
Assay Kit
Cat. No. ECM640
Sufficient for the analysis of 24 samples
FOR RESEARCH USE ONLY
Not for use in diagnostic procedures
USA & Canada
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Introduction
A fundamental requirement for the physiological performance of organs is the
formation of diffusion barriers that separate and maintain compartments of
different structure. The endothelial cell lining of the internal vasculature defines
a semi-permeable barrier between the blood and the interstitial spaces of the
body. This barrier is composed of intracellular adherent, tight, and gap junction
complexes, as well as desmosomes1. Junction substructure components such as
connexins, integrins, cadherins, catenins, occludins, desmoplakins, selectins,
and PECAM-1 all act as interface regulators for paracellular permeability of
nutrients, chemicals, and ions2,3. Endothelial cell adhesive characteristics
provide strength and stability for neighboring cells and the cellular cytoskeleton
by interacting with actin and myosin contractile filaments4,5. Junctional
molecules also influence cell signaling and trigger responses that are translated
into cell morphology changes and physiological angiogenesis6,7.
A multitude of vasoactive cytokines, growth factors, and signal modulators react
with endothelial cell substructure components to control permeability. VEGF,
Interleukin alpha and beta, TNF alpha, and IFN gamma have been shown to
increase endothelial monolayer permeability8,9,10,11. Thrombin stimulation of
cytoskeletal signaling pathways has been shown to manipulate cell
permeability12. Lipopolysaccharide (LPS) induces junction barrier loss and cell
detachment by activating PTKs and caspase cleavage reactions13. In contrast,
junctional adhesion molecule (JAM) decreases permeability by initiating cell
adhesion14.
Disruptions of the barrier integrity are manifested as microvascular
hyperpermeability, which is associated with many systemic disease states.
Pathological angiogenic disease states include heart disease, diabetes, cancer,
stroke, hypertension, arthritis, and Alzheimer’s1,15,16. Increases in tissue
permeability may be caused by weak, hemorrhaging vessels that become
oedematous, and intensifies with irregular fluid flow through the vessels15.
Expanding the knowledge of endothelial junction behavior and the agents that
influence that behavior will lead to new therapies for controlling endothelial
permeability.
An essential ingredient of any in vitro permeability study is an intact, confluent
cell monolayer. Endothelial cell monolayers cultured on semi-permeable
membranes have been shown to form adherent and tight junctions17. The
CHEMICON In Vitro Vascular Permeability Assay Kit provides an efficient
system for evaluating the effects of chemicals and drugs on endothelial cell
adsorption, transport, and permeability.
1
Test Principle
The CHEMICON® In Vitro Vascular Permeability Assay is performed in a 24well tissue culture plate with 12 cell culture inserts. The inserts contain 1.0 µm
symmetrical pores within a transparent polyethylene membrane. The high pore
density membranes permit amplified rates of basolateral diffusion with
molecules of interest for permeability assays. The membrane is tissue culture
treated on both sides for cell growth.
Within CHEMICON®’S In Vitro Vascular Permeability Assay, endothelial cells
are seeded onto collagen-coated inserts. The endothelial monolayer forms in
several days, which occludes the membrane pores. The cell monolayer is then
treated with cytokines, growth factors, or some reagent of interest. After
treatment, FITC-Dextran is added on top of the cells, allowing it to permeate
through the cell monolayer. The extent of permeability can be determined by
measuring the fluorescence of the plate well solution.
2
Application
The CHEMICON® In Vitro Vascular Permeability Assay Kit is ideal for
measuring compounds that disrupt an endothelial monolayer.
Each
®
CHEMICON In Vitro Vascular Permeability Assay Kit contains sufficient
reagents for the evaluation of 24 samples. The CHEMICON® In Vitro Vascular
Permeability Assay Kit is intended for research use only, not for diagnostic or
therapeutic applications.
Storage
Store kit materials at 2-8°C for up to 12 months. Do not freeze.
3
Kit Components
1.
Permeability Chamber: (Part No. 90326) Two 24-well plates with 12 cell
culture inserts per plate (24 inserts total/kit).
2.
Permeability Detection Plate: (Part No. 90329) One 24-well plate.
3.
Collagen Solution: (Part No. 90327) One bottle – 1.0 mL.
4.
FITC-Dextran Solution: (Part No. 90328) One vial – 0.25 mL.
5.
Forceps: (Part No. 10203) One set of forceps.
Materials Not Supplied
1.
Cell Basal Medium
2.
Cell Growth Medium
3.
Harvesting Buffer: EDTA or trypsin cell detachment buffer
4.
Sterile PBS or HBSS (Hank’s Balanced Salt Solution) for washing cells
5.
Sterile pipettes and pipette tips sufficient for aliquoting cells
6.
Low speed centrifuge and tubes for cell harvesting
7.
CO2 tissue culture incubator
8.
Microscope
9.
Hemacytometer
10. Trypan blue or equivalent viability stain
11. Fluorescence plate reader with 485nm and 530nm filters
12. 96 well fluorescence black plate or other suitable plate
13. Sterile cell culture hood
14. Vascular Permeability Factor (e.g. VEGF, IL-1α, IL-1β, TNFα, etc.)
15. Sterile PBS, pH 7.1
16. Sterile 0.1N NaOH
17. Sterile deionized water
4
Preparation of Reagents
1.
Collagen Coating Solution
Under sterile conditions, prepare the required volume of 0.2X PBS (see
table below) by diluting 1X PBS (pH 7.1) 1:5 with deionized water. Mix
solution thoroughly, and cool to 2-8˚C. Next, add the required Collagen
Solution to the pre-chilled 0.2X PBS solution. Vortex solution thoroughly.
Number
of
Inserts
Collagen
Solution
Required
0.2X PBS
Required
Total Volume of
Collagen Coating
Solution
6
35 µL
2.465 mL
2.5 mL
12
70 µL
4.93 mL
5 mL
24
140 µL
9.86 mL
10 mL
Finally, adjust the pH of the collagen coating solution to 7.0 ± 0.1 with
sterile 0.1N NaOH, verifying by pH paper (Note: ~20 µL of 0.1N NaOH
per 10 mL of collagen solution). Mix to homogeneity. Use the mixture
immediately (see Assay Instructions).
2.
FITC-Dextran
Prepare a working solution of the FITC-Dextran with basal media. A
dilution of 1:40 is suggested, although the final dilution may need to be
optimized by the end user.
Cell Harvesting
Perform the following steps in a sterile hood
Prepare cell line for investigation as desired. The following procedure is a
suggestion for preparing endothelial cells used in monolayer formation.
1.
Use cells that are at least 80% 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
5.
Add 10-20 mL of 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 (1500 RPM, 5-10 minutes).
7.
Gently resuspend the pellet in 1-5 mL of Cell Growth Medium, depending
upon the size of the pellet.
8.
Count cells and bring to a volume that provides 1.0 –2.0 x 106 cells per mL.
9.
Add additional compounds (cytokines, pharmacological agents, etc.) to the
cell suspension if pretreatment of the endothelial cells is necessary.
Assay Instructions
Perform the following steps in a sterile hood
1.
Sterilize forceps with 70% ethanol and only handle inserts with forceps.
2.
Under a sterile hood, add 200 µL of the collagen coating solution (see
Preparation of Reagents section) to each insert being assayed. Make sure
that the solution completely covers the bottom of the insert.
3.
Cover the plate and incubate at room temperature for 1 hour.
4.
Under sterile conditions, remove the solution from the inserts by inverting
and shaking them in a waste container. Return the inserts to the plate.
5.
Hydrate the inserts with 250 µL of Cell Growth Medium. Make sure that
the solution completely covers the bottom of the insert.
6.
Cover plate and incubate at room temperature for 15 minutes.
7.
Prior to adding cells, remove 200 µL of the Cell Growth Medium solution
from the insert and discard.
8.
Seed 200 µL of the cell suspension to the insert. (Example: HUVECs at
1.0 x 106 cells/mL).
9.
Carefully add 500 µL of Cell Growth Medium to the plate well.
Note: Ensure that the media completely covers the underside of the insert
and that no bubbles are present. Air may get trapped at the interface.
10. Cover the plate and incubate the cells for 72 hours, or until a monolayer is
formed, in a 37˚C CO2 tissue culture incubator.
6
11. If a cell starvation step is not necessary, proceed to step 13. If a cell
starvation step is necessary, replace the media in the insert and plate well
with starvation media (e.g. Cell Basal Medium + 0.5% FBS).
Note: This step should be done quickly, one at a time, to ensure the cell
monolayer does not dry.
12. Cover the plate and incubate the cells 6 to 24 hours in a 37˚C CO2 tissue
culture incubator.
13. Carefully remove the media from the insert and the plate so as not to touch
or disturb the monolayer.
14. Transfer the insert to a fresh plate well.
15. Add the vascular permeability factor in 200 µL of Cell Basal Medium to the
inserts being tested. Add 500 µL of the solution to the plate wells.
16. Cover the plate and incubate the cells 4 to 18 hours as necessary in a 37˚C
CO2 tissue culture incubator.
17. Again, carefully remove the media from the insert and the plate so as not to
touch or disturb the HUVEC monolayer.
18. Transfer the inserts to the Permeability Detection Plate.
19. Add 500 µL of Cell Basal Medium to the bottom plate well for all inserts.
20. Add 150µL of FITC-Dextran in Cell Basal Medium to each insert.
Incubate the plate for 5 minutes at room temperature.
21. Stop the reaction by removing the inserts from the wells. Mix the plate well
solution thoroughly.
22. Remove 100 µL of the plate well solution and transfer it to a 96-well plate
suitable for fluorescence measurement.
23. Read the plate using a fluorometer with a 485 nm and 530 nm filter set.
Samples can be returned to their respective plate wells, and the inserts
replaced for subsequent time point readings.
7
Calculation of Results
Results of the ECM 24-well In Vitro Permeability Assay may be illustrated
graphically by the use of a “bar” chart. HUVEC monolayers treated with
endothelial cell growth media only were used as positive controls for monolayer
integrity as indicated by the low fluorescence values for the FITC-Dextran.
Fluorescence measurements were determined on the CytoFluor 4000 plate
reader (Perceptive Biosystems) using excitation/emission wavelengths of
485nm/530nm and a Gain setting of 65. The Gain setting may be adjusted for
optimal fluorescence.
The data below should be used for reference only. This data should not be used
to interpret actual assay results.
HUVEC Monolayer Permeability
Relative Fluorescence Units (RFUs)
50000
40000
30000
20000
10000
0
Basal Medium
Only
IL-1beta
Growth Medium
Only
No Cell
Monolayer
Samples Tested
Figure 1: HUVECs were seeded at 200,000 cells per insert and cultured for 72
hours. HUVEC monolayer permeability tested after a 24 hour starvation period.
Test inserts were then treated for 18 hours with 100 ng/mL of Interleukin-1β in
basal medium. Monolayers were also treated with basal medium and growth
medium only. Inserts were also tested without cell monolayer.
8
References
1.
Bingmei M. Fu. Microvessel Permeability and Its Regulation. Recent
Advances in Biomechanics 231-247, Springer-Verlag, Berlin Heidelberg,
2001.
2.
Navarro P. et al. (1995). Catenin-dependent and -independent Functions of
Vascular Endothelial Cadherin. Journal of Biological Chemistry 270:
30965-30972.
3.
Dejana E. (1996). Endothelial Adherens Junctions: Implications in the
Control of Vascular Endothelial Cadherin (cadherin-5/VE-cadherin).
Journal of Clinical Investigation 98: 886-893.
4.
Schnittler H.J. (1990). Role of Actin and Myosin in the Control of
Paracellular Permeability in Pig, Rat, and Human Vascular Endothelium.
Journal of Physiology 431: 379-401.
5.
Dudek S. M., et al. (2001). Cytoskeletal Regulation of Pulmonary
Vascular Permeability. Journal of Applied Physiology 91: 1487-1500.
6.
Krizbai I.A. et al. (2003). Signalling Pathways Regulating the Tight
Junction Permeability in the Blood-Brain Barrier. Molecular Biology of the
Cell 49: 23-31.
7.
Dejana E. (1996). Endothelial Adherens Junctions: Implications in the
Control o Vascular Permeability and Angiogenesis. Journal of Clinical
Investigation 98: 1949-1953.
8.
Lal B.K. et al. (2001). VEGF Increases Permeability of the Endothelial
Cell Monolayer by Activation of PKB/akt, Endothelial Nitric-Oxide
Synthase, and MAP Kinase Pathways. Microvascular Research 62: 252262.
9.
Burke-Gaffney A., et al. (1993). Modulation of Human Endothelial Cell
Permeability by Combinations of the Cytokines Interleukin-1 Alpha/Beta,
Tumor
Necrosis
Factor-Alpha
and
Interferon-Gamma.
Immunopharmacology 25: 1-9.
10. Marcus B.C., et al. (1996). Cytokine-Induced Increases in Endothelial
Permeability Occur After Adhesion Molecule Expression. Surgery 120:
411-417.
11. Campbell W.N., et al. (1992). Interleukin-1 Alpha and –Beta Augment
Pulmonary Artery Transendothelial Albumin Flux In Vitro. American
Journal of Physiology 263: 128-136.
9
12. Vouret-Craviari V., et al. (1998). Regulation of the Actin Cytoskeleton by
Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial
Barrier Function. Molecular Biology of the Cell 9: 2639-2653.
13. Bannerman D.D., et al. (1998). Bacterial Lipopolysaccharide Disrupts
Endothelial Monolayer Integrity and Survival Signaling Events Through
Caspase Cleavage of Adherens Junction Proteins. Journal of Biological
Chemistry 273: 35371-35380.
14. Bazzoni G., et al. (2000). Interaction of Junctional Adhesion Molecule
with the Tight Junction Components ZO-1, Cingulin, and Occludin.
Journal of Biological Chemistry 275: 20520-20526.
15. Bates D.O., et al. (2002). Regulation of Microvascular Permeability by
Vascular Endothelial Growth Factors. Journal of Anatomy 200: 581-597.
16. Mooradian A.D. (1988). Effect of Aging on the Blood-Brain Barrier.
Neurobiological Aging 9: 31-39.
17. Esser S., et al. (1998). Journal of Cell Science 111: 1853.
10
Warranty
These products are warranted to perform as described in their labeling and in
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.
2003: 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.
Cat. No. ECM640
December 2003
Revision A: 41617