ECM644 In Vitro Vascular Permeability Assay (24-well)

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ECM644
24 assays  
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      Overview

      Replacement Information
      Replacement InformationECM640
      NOW AVAILABLE!
      96-well (ECM642) Collagen Pre-coated Version

      Key Specifications Table

      Detection Methods
      Fluorescent
      Description
      Catalogue NumberECM644
      ReplacesECM640
      Brand Family Chemicon®
      Trade Name
      • Chemicon
      DescriptionIn Vitro Vascular Permeability Assay (24-well)
      OverviewNOW AVAILABLE!
      In Vitro Vascular Permeability Imaging Assay (17-10398)

      Read our application note in Nature Methods!
      http://www.nature.com/app_notes/nmeth/2012/121007/pdf/an8623.pdf
      (Click Here!)

      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 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 manipulate cell permeability. Lipopolysaccharide (LPS) induces junction barrier loss and cell detachment by activating protein tyrosine kinases (PTKs) and caspase cleavage reactions. 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.

      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 junctions. The Millipore In Vitro Vascular Permeability Assay kit provides an efficient system for evaluating the effects of chemicals and drug compounds on endothelial cell adsorption, transport, and permeability.

      Test Principle:

      The Millipore In Vitro Vascular Permeability Assay is performed in a 24-well receiver plate with 24 individual hanging cell culture inserts. The inserts contain 1 µm pores within a transparent polyethylene terephthalate (PET) membrane. Each insert has been pre-coated with an optimized concentration of type I rat-tail collagen. The high pore density membranes permit apical and basolateral access of cells to media and permeability molecules of interest.

      Within Millipore’s In Vitro Vascular Permeability Assay, endothelial cells are seeded onto the collagen-coated inserts. An endothelial monolayer forms in several days, which occludes the membrane pores. The cell monolayer is then treated with cytokines, growth factors, or other compounds of interest. After treatment, a high molecular weight FITC-Dextran is added on top of the cells, allowing the fluorescent molecules to pass through the endothelial cell monolayer at a rate proportional to the monolayer’s permeability. The extent of permeability can be determined by measuring the fluorescence of the receiver plate well solution.
      Materials Required but Not Delivered1. Human umbilical vein endothelial cells (HUVEC) such as EndoGRO™ Human Umbilical Vein Endothelial Cells (Cat. No. SCCE001) or endothelial cell type of interest.
      2. Endothelial cell Basal Medium. (Note: Phenol red should be avoided—it may interfere with fluorescence measurement and increase background fluorescence.)
      3. Endothelial cell Growth Medium.
      4. Vascular permeability factor (e.g., IL-1β, TNF-α, VEGF, Ang-1, etc.).
      5. Cell detachment buffer (e.g., 0.05% trypsin).
      6. Sterile phosphate-buffered saline (PBS) or Hank’s balanced salt solution (HBSS) for washing of cells.
      7. Sterile cell culture hood.
      8. Pipettors, liquid aspirators, etc. for handling of cells and liquid reagents.
      9. Sterile plasticware (cell culture flasks, centrifuge tubes, pipettes, pipette tips, etc. for handling of cells).
      10. CO2 tissue culture incubator.
      11. 70% ethanol for disinfection of forceps, plasticware, surfaces, etc.
      12. Low speed centrifuge for cell harvesting.
      13. Hemocytometer.
      14. Trypan blue or equivalent viability stain.
      15. Microscope (for phase contrast and brightfield imaging).
      16. Fluorescence plate reader with filters for 485 nm and 535 nm excitation and emission, respectively (appropriate for FITC/fluorescein signal).
      References
      Product Information
      Components
      • 24-Well Permeability Insert Assembly, Collagen-coated: One 24-well receiver tray containing 24 porous cell culture inserts pre-coated with type I rat-tail collagen.
      • Receiver Tray, 24-Well: Two 24-well plates.
      • FITC-Dextran Solution: One vial containing 250 µL.
      • Forceps: One set of forceps.
      • Cell Stain: One bottle containing 10 mL.
      • Black Plate, 96-Well Opaque: One 96-well plate.
      Detection methodFluorescent
      Quality LevelMQ100
      Applications
      ApplicationThis In Vitro Vascular Permeability Assay kit employs a 24-well plate and provides an efficient system for evaluating the effects of chemicals & drug compounds on endothelial cell adsorption, transport & permeability.
      Application NotesThe Millipore® In Vitro Vascular Permeability Assay kit provides an efficient system for evaluating the effects of chemicals and drug compounds on endothelial cell adsorption, transport, and permeability. It is ideal for measuring compounds that may disrupt or protect an endothelial monolayer. Each In Vitro Vascular Permeability Assay Kit contains sufficient reagents for the evaluation of 24 samples. The In Vitro Vascular Permeability Assay Kit is intended for research use only, not for diagnostic or therapeutic applications.
      Biological Information
      Species Reactivity
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      Physicochemical Information
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      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage ConditionsStore kit materials at 2-8°C; use within 4 months from date of receipt. Do not freeze.
      Packaging Information
      Material Size24 assays
      Transport Information
      Supplemental Information
      Specifications

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      Categories

      Life Science Research > Cell Analysis > Cell-based Assays > Angiogenesis & Endothelial Transmigration Assays