Key Specifications Table
|Description||QCM™ High Sensitivity Non-cross-linked Collagen Invasion Assay, 24-well (8 µm), Colorimetric|
|Overview||Also available: Cell Comb™ Scratch Assay! Get biochemical data from a scratch assay! Click Here
Metastasis, the process by which tumor cells colonize tissues distant from the site of the primary tumor, consists of multiple phases: invasion of cells from the primary tumor into adjacent tissues, passage of the tumor cells through the vascular lining into the bloodstream (intravasation), adhesion to the endothelium at the site of implantation, traversal across the endothelium (extravasation) and proliferation at the distant site. The first step, invasion, requires that the tumor cell traverse one or more extracellular matrix elements, depending on the tumor type. Epithelial tumors must breach a basement membrane, which is a dense sheet of interwoven cross-linked type IV collagen and laminin, in order to extend into neighboring tissues. In addition, many tumors contain a reactive stroma containing fibrillar collagen, another cross-linked matrix element. Finally, the process of intravasation requires passage through the endothelial basement membrane.
The mechanisms by which premetastatic cells breach such barriers are the focus of intense study. In many solid tumors of epithelial origin, a morphological change from a cuboidal epithelial phenotype to a spindle-shaped mesenchymal phenotype (termed the epithelial-mesenchymal transition, or EMT) presages the invasion of the cells across ECM barriers. A key feature of the EMT is the increased expression of proteases, chiefly matrix metalloproteinases, which cleave the components of the ECM. However, alternative forms of cell invasion have been observed and are likely to contribute to the invasive potential of a tumor. Some invasive cells adopt an amoeboid phenotype, and migrate by rear-force propulsion and membrane blebbing; such amoeboid cells invade the ECM by squeezing through pores and appear not to require proteases. Distinctions between amoeboid and mesenchymal invasion are not clear-cut, and some tumor cells will adopt different morphologies in response to environmental conditions.
The most widely used in vitro assay to assess invasive potential of a cell type employs a chamber with a porous filter at the bottom, which is coated with a gelled layer of ECM, usually collagen or basement membrane extract. Cells are applied to the top of the gel, and a chemoattractant is provided in the well below the chamber, such that invasive cells traverse the ECM and move through the pores to the other side, where they are then quantified. EMD Millipore provides such assays, coated with either basement membrane extract (Catalogue No. ECM550, ECM554, ECM555) or collagen (Catalogue No. ECM551, ECM552, ECM556).
As our understanding of migration modes expands, more consideration is being given to the properties of the ECM employed in such in vitro models of invasion, and the extent to which they reflect the properties of the ECM of the tumor microenvironment. Basement membrane extracts contain the constituents, but lacks the covalent cross-links, of a basement membrane in vivo. Two forms of fibrillar collagen are used in invasion assays. Acid-extracted collagen, which is employed in EMD Millipore’s Collagen Invasion Assays (Catalogue No. ECM551, ECM552, ECM556), retains intermolecular covelent cross-links, whereas pepsin-solubilized collagen, also known as atelo-collagen, lacks such cross-links. It has been demonstrated that amoeboid invasion only occurs in matrix environments lacking cross-links.
EMD Millipore’s QCM™ High Sensitivity Non-cross-linked Collagen Invasion Assay utilizes the same format of matrix-coated porous filters as the existing products, but employs a non-cross-linked, atelo-collagen that provides a more pliable environment that permits higher levels of cell invasion to occur. For moderately invasive cell types, invasion can be detected at earlier time points with the High Sensitivity Collagen Invasion Assay. In less invasive cell types, the High Sensitivity Collagen Invasion Assay is able to detect invasion that is not apparent with the original QCM™ Collagen Invasion Assay.
|Materials Required but Not Delivered||• Precision pipettes sufficient for aliquoting cells
• Sterile cell culture hood
• CO2 incubator appropriate for subject cells
• Tissue culture growth medium appropriate for subject cells, such as DMEM 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
• Quenching Medium: serum-free medium containing 5% BSA (for EDTA/PBS) or soybean trypsin inhibitor (for trypsin)
• Sterile PBS or HBSS to wash cells
• 70% ethanol
• Distilled water
• Low speed centrifuge and tubes for cell harvesting
• Hemocytometer or other means of counting cells
• Trypan blue or equivalent viability stain
• Chemoattractants (eg. 10% FBS) or pharmacological agents for addition to culture medium, if screening is desired
• Microplate reader (560 nm)
• 24-well tissue culture plate
• 96-well plate
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Store kit materials at 2-8°C for up to 4 months from date of receipt. Do not freeze.|
|Material Size||24 wells|
|Material Package||24 wells|
QCM™ High Sensitivity Non-cross-linked Collagen Invasion Assay, 24-well (8 µm), Colorimetric SDS
|QCMTM High Sensitivity Non-cross-linked Collagen Invasion Assay, 24-well (8 μm) Colorimetric|