Key Specifications Table
|Description||QCM Collagen Cell Invasion Assay, 24-well (8 µm), Fluorometric|
|Overview||Also available: Cell Comb™ Scratch Assay! Get biochemical data from a scratch assay! Click Here
Penetration of the subendothelial basement membrane marks a critical turning point in the metastatic process. As proliferating neoplastic cells attempt to escape the primary tumor site, local invasion of the surrounding tissue (interstitial stroma) must occur. Neovascularization is initiated by expression of angiogenic factors (e.g. FGF, VEGF, HGF), providing nutritional requirements and access to the vascular system. Prior to penetrating the blood vessel endothelium and gaining access to the blood stream (intravasation), cancer cells must invade local tissues by degrading ECM components and ultimately, transverse the basement membrane. Once in circulation, these cells can form metastatic colonies at secondary locations, making this membrane a key invasive barrier.
The basement membrane surrounding the blood vessel endothelium is a thin, specialized network of extracellular matrix proteins (ECM) that serves many functions. Comprised of proteins and proteoglycans, such as collagen, laminin, entactin, fibronectin, heparin sulfate and perlecan, this membrane acts as a physical barrier between the epithelium and underlying tissues. It provides cell surface anchorage (via integrins, receptor kinases, and cell surface proteoglycans), induces cellular differentiation, gives architectural support, and limits the migration of normal cells. The ability of tumor cells to degrade the ECM components of the basement membrane and surrounding tissues is directly correlated with metastatic potential. By releasing proteolytic enzymes (e.g. MMP collagenases, plasminogen activators, cathepsins), cancer cells are able to breach the membrane and penetrate the blood vessel wall (1). Collagen, the primary structural element of the basement membrane and tissue scaffolding protein, represents the main deterrent in the migration of tumor cells.
The ability to study cell invasion through a collagen barrier, is of vital importance for developing possible metastatic inhibitors and therapeutics. The new CHEMICON QCM™ 24-well Collagen-based Invasion Assay (ECM552) provides an efficient, in vitro system for quantitative analysis of tumor cell invasion.
In the QCM™ 24-well Collagen-based Invasion Assay (ECM552), invaded cells on the bottom of the insert membrane are dissociated from the membrane when incubated with a specially formulated Cell Detachment Buffer. The invasive cells are subsequently lysed and detected by the patented CyQuant GRÃÂ¢ dye (Molecular Probes) (2-3). This green-fluorescent dye exhibits strong fluorescence enhancement when bound to cellular nucleic acids (4).
The CHEMICON QCM™ 24-well Collagen-based Invasion Assay (ECM552) eliminates cell pre-labeling, fixing/staining, swabbing, and manual counting. The 24-well insert and homogenous fluorescence detection format allows for quantitative comparison of multiple samples.
In addition, Chemicon continues to provide numerous migration, invasion, and adhesion products including:
The CHEMICON Cell Invasion Assay is performed in an Invasion Chamber, based on the Boyden chamber principle. Each kit contains 24 inserts; each insert contains an 8 mm pore size polycarbonate membrane coated with a thin layer of polymerized avian collagen. The collagen layer occludes the membrane pores, blocking non-invasive cells from migrating through. Invasive cells, on the other hand, migrate through the polymerized collagen layer and cling to the bottom of the polycarbonate membrane. Invaded cells on the bottom of the insert membrane are dissociated from the membrane when incubated with Cell Detachment Buffer and subsequently lysed and detected by CyQuant GR dye.
|Materials Required but Not Delivered||1. Precision pipettes: sufficient for aliquoting cells.
2. 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.
Note: Trypsin cell detachment buffer maybe required for difficult cell lines. Allow sufficient time for cell receptor recovery.
3. Tissue culture growth medium appropriate for subject cells, such as DMEM containing 10% FBS.
4. Chemoattractants (eg. 10% FBS) or pharmacological agents for addition to culture medium, if screening is desired.
5. Quenching Medium: serum-free medium, such as DMEM, EMEM, or FBM (fibroblast basal media), containing 5% BSA.
Note: Quenching Medium must contain divalent cations (Mg2+, Ca2+) sufficient for quenching EDTA in the harvesting buffer.
6. Sterile PBS or HBSS to wash cells.
7. Distilled water.
8. Low speed centrifuge and tubes for cell harvesting.
9. CO2 incubator appropriate for subject cells.
10. Hemocytometer or other means of counting cells.
11. Trypan blue or equivalent viability stain.
12. Fluorescence plate reader.
13. Sterile cell culture hood.
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Store kit materials at 2-8°C for up to their expiration date. Do not freeze.|
|Material Size||1 plate|
|Material Package||24 wells|