Key Specifications Table
|Description||In Vitro Vascular Permeability Imaging Assay (Green)|
96-well ( ECM642) & 24-well ( ECM644) Collagen Pre-coated Versions
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 intercellular adherens, tight, and gap junction complexes, as well as desmosomes. Junction substructure components such as connexins, integrins, cadherins, catenins, occludins, desmoplakins, selectins, and platelet endothelial cell adhesion molecule-1 (PECAM-1) all act as interface regulators for paracellular permeability of ions, nutrients, therapeutic agents, and macromolecules. Endothelial cell adhesive characteristics provide strength and stability for neighboring cells and the cellular cytoskeleton by interacting with actin and myosin contractile filaments. Junctional molecules also influence cell signaling and trigger responses that are translated into cell morphology changes and physiological angiogenesis.
A multitude of vasoactive cytokines, growth factors, and signal modulators react with endothelial cell substructural components to control permeability. Vascular endothelial growth factor (VEGF), interleukin-1 alpha and beta (IL-1α and IL-1β), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) have been shown to increase endothelial monolayer permeability. Thrombin stimulation of cytoskeletal signaling pathways has been shown to increase cell permeability. In contrast, junctional adhesion molecule (JAM) decreases permeability by initiating cell adhesion and angiopoietin-1 (Ang-1) can protect endothelial barrier function through regulation of junctional complexes.
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’s. Increases in tissue permeability may be caused by weak, hemorrhaging vessels that become oedematous, and intensifies with irregular fluid flow through the vessels. Expanding the knowledge of endothelial junction behavior and the agents that influence that behavior will lead to new therapies for controlling endothelial permeability.
EMD Millipore’s In Vitro Vascular Permeability Imaging Assay provides optimized materials and protocols to enable detailed spatial analysis of intercellular permeability in endothelial monolayers. All of the components necessary for affixing a thin film of biotinylated matrix to glass culture surfaces and staining sites of intercellular permeability are provided. In addition, compatible reagents are provided for correlating tight junctions (VE-cadherin) and nuclei with sites of permeability. This assay may be used for assessing activity of inhibitors and promoters of vascular permeability, and correlating permeability with ultrastructural features and signaling events.
The EMD Millipore In Vitro Vascular Permeability Imaging Assay provides the reagents necessary for affixing a thin, uniform layer of biotinylated gelatin to a glass culture substrate which, in the presence of an endothelial monolayer, binds to a fluorescently labeled streptavidin only at sites of intercellular permeability. A poly-L-lysine coating is first adsorbed to the glass substratum. The substrate is then treated with a dilute glutaraldehyde solution to bi-functionally “activate” the surface for further protein binding. Subsequent incubation of the surface with biotinylated gelatin allows covalent coupling between the poly-L-lysine and gelatin via reactive aldehyde (-CHO) groups. The biotin-coated glass is now prepared for cell culture by disinfection with 70% ethanol, followed by quenching of free aldehydes with amino acid-containing growth medium.
The endothelial cell of interest is seeded onto the gelatin surface and allowed to form a confluent monolayer. Treatment compounds of interest may be introduced at desired time points during the culture period. Sites of intercellular permeability are then stained with fluorescent streptavidin , which are microscopically visualized and may be quantified using image analysis software algorithms. The assay also provides anti-VE-cadherin and DAPI, for visualization of adherens junctions and nuclei, respectively, to allow colocalization of sites of adherens junction remodeling and increased permeability. The basic method allows potential activators or inhibitors to be investigated for their influence on the degree and sites of vascular permeability. The assay may be further combined with immunocytochemical staining for other molecules of interest to colocalize sites of permeability with signaling events.
|Materials Required but Not Delivered||1. Sterile cell culture hood
2. Pipettors, liquid aspirators, etc. for handling of cells and liquid reagents
3. Sterile plasticware (cell culture flasks, centrifuge tubes, pipettes, pipette tips, etc. for handling of cells and liquid reagents)
4. Sterile glass substrate (e.g., chamber slide, coverslip, glass-bottom dish/multi-well plate)
5. Sterile deionized water
6. Sterile Dulbecco’s phosphate-buffered saline (DPBS), without calcium or magnesium
7. 70% ethanol in sterile water
8. Human umbilical vein endothelial cells (HUVEC) such as EndoGRO™ Human Umbilical Vein Endothelial Cells (Cat. No. SCCE001) or endothelial cell type of interest
9. Endothelial cell Basal Medium
10. Endothelial cell Growth Medium
11. Vascular permeability factor (e.g., IL-1β, TNF-α, VEGF, Ang-1, thrombin etc.)
12. Hemocytometer (e.g. Scepter™ Handheld Automated Cell Counter)
13. Trypan blue or equivalent viability stain
14. Low speed centrifuge for cell harvesting
15. CO2 tissue culture incubator
16. 3.7% formaldehyde in DPBS (or equivalent) for cell fixation
17. Blocking/permeabilization buffer for antibody/DAPI staining (e.g., 2% blocking serum/0.25% Triton X-100 in DPBS)
18. Slide mounting media (with anti-fade reagent) and cover glasses, if appropriate
19. Microscope/image acquisition system (for phase contrast and fluorescence)
20. Fluorescence filters for fluorescein, Cy3 and DAPI imaging (see Table 2 for specific excitation/emission wavelengths)
21. Image analysis software (e.g., NIH ImageJ)
|Safety Information according to GHS|
|Material Size||32 assays|
In Vitro Vascular Permeability Imaging Assay (Green) SDS
|In Vitro Vascular Permeability Imaging Assay (Green)|