Key Specifications Table
|Description||QCM Collagen Cell Invasion Assay, 96-well (8 µm), fluorimetric|
|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™ 96-well Collagen-based Invasion Assay (ECM556) provides an efficient, in vitro system for quantitative, high-throughput analysis of tumor cell invasion.
In the QCM™ 96-well Collagen-based Invasion Assay (ECM556), 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 dye (Molecular Probes) (2-3). This green-fluorescent dye exhibits strong fluorescence enhancement when bound to cellular nucleic acids (4).
The CHEMICON QCM™ 96-well Collagen-based Invasion Assay (ECM556) eliminates cell pre-labeling, fixing/staining, swabbing, and manual counting. The 96-well insert and homogenous fluorescence detection format allows for high-throughput, 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 a 96-well invasion plate based on the Boyden chamber principle. This plate contains 96 inserts; each insert contains an 8 μm pore size polycarbonate membrane coated with a thin layer of polymerized 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||96 wells|
|Reference overview||Pub Med ID|
|A conjugate of camptothecin and a somatostatin analog against prostate cancer cell invasion via a possible signaling pathway involving PI3K/Akt, alphaVbeta3/alphaVbeta5 and MMP-2/-9.|
Li-Chun Sun, Jing Luo, L Vienna Mackey, Joseph A Fuselier, David H Coy
Cancer letters 246 157-66 2007
Camptothecin (CPT) was conjugated to the N-terminal of a somatostatin analog (SSA) directly via a carbamate group and a basic N-terminal linking motif, D-Lys-D-Tyr-Lys-D-Tyr-D-Lys. This new CPT-SSA conjugate termed JF-10-81 was evaluated as a receptor-specific delivery system for its anti-invasive and anti-angiogenic activities. It was found that, in addition to blocking migration and invasion of highly invasive prostate cancer PC-3 cells, this conjugate also inhibited in vitro capillary-like tube formation of endothelial cells and in vivo angiogenesis in C57B1/6N female mice. JF-10-81 was found to block PC-3 cell attachment to various extracellular matrix components, mainly to vitronectin, the ligand of cell surface receptors integrin alphaVbeta3 and alphaVbeta5. Additionally, JF-10-81 reduced expression of integrins alphaVbeta3 and alphaVbeta5 on PC-3 cell surfaces, without effects on beta1 or any alphabeta1 heterodimers. This conjugate also inactivated phosphorylation of protein kinase B (PKB/Akt), down-regulated the expression of latent matrix metalloproteinase (MMP) -2 and MMP-9, but had little effect on MMP-3/-10. Meanwhile, membrane type-1 matrix metalloproteinase (MT1-MMP) and the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) were not detectable in PC-3 cells. alphaVbeta3/alphaVbeta5 and MMP-2/-9 are known to be highly expressed in many tumor cells and play an important role in tumor progression. Our results support that this conjugate could possibly inhibit prostate cancer PC-3 cell invasion through a signaling pathway involving PI3K/Akt, alphaVbeta3/alphaVbeta5 and MMP-2/-9, and this SSA could be used as an efficient vector to deliver CPT or other cytotoxic agents to target sites for cancer therapy.
|Effects of camptothecin on tumor cell proliferation and angiogenesis when coupled to a bombesin analog used as a targeted delivery vector.|
Li-Chun Sun,Jing Luo,Vienna L Mackey,Joseph A Fuselier,David H Coy
Anti-cancer drugs 18 2007
The camptothecin-bombesin conjugate termed DC-51-43, as a novel targeted drug delivery system, was examined in over 10 human tumor cell lines and shows a potent antiproliferative activity. This conjugate has also demonstrated its antitumor activity in our previous experiments. In our present study, we evaluate this conjugate for its antiangiogenic activity by in-vitro and in-vivo experiments. The camptothecin-bombesin conjugate and free camptothecin show potent in-vitro inhibitory activities of cell adhesion to various extracellular matrix components and integrins alphaVbeta3 and alphaVbeta5, not beta1/alphabeta1. This conjugate displays inhibitory activity to cell migration and invasion at concentrations of 10 micromol/l or above. This conjugate is also effective against in-vitro capillary-like tube formation of endothelial cells (at 40 micromol/l), and in-vivo angiogenesis as seen by blocking the spread of host mice endothelial cells into matrigel plugs. These experimental results support the fact that the camptothecin-bombesin conjugate has therapeutic activities against angiogenesis. By binding to bombesin receptor-expressing sites, this bombesin analog, consisting of 11 amino acids, is potentially a novel delivery vector for nonspecific cytotoxic agents.
|Advancing cancer research: From hallmarks & biomarkers to tumor microenvironment progression|
|Cell Migration and Invasion: Choosing the Right Assay|
|QCM Collagen Cell Invasion Assay, Fluorimetric - Data Sheet|
|QCM Collagen Cell Invasion Assay, Fluorimetric - Data Sheet|