Key Spec Table
|Description||QCM 3 µm Endothelial Cell Migration Assay Fibronectin, Colorimetric|
|Overview||Also available: Cell Comb™ Scratch Assay! Get biochemical data from a scratch assay! Click Here
Angiogenesis is a fundamental process involving the growth of new blood vessels from pre-existing vessels. It is important in development and wound healing, as well as pathologic diseases such as diabetic retinopathy and cancer. During angiogenesis, endothelial cells need to move out of existing vessels, migrate into new areas, proliferate and assemble into new capillaries. The migration of endothelial cells is regulated by many angiogenic factors and anti-angiogenic factors. It is critical for researchers to understand the mechanisms of endothelial cell migration.
Millipore's 3 μm QCM™ Endothelial Cell Migration Assay – Fibronectin, Colorimetric provides a quick and efficient system to study the ability of compounds to induce or inhibit endothelial cell migration. This assay also allows screening of pharmacological agents, evaluation of integrins or other adhesion receptors responsible for endothelial cell migration, analysis of gene function in transfected cells, and determination of ECM protein involvement in cell movement.
This versatile assay permits counting of individual migratory cells, and, more importantly, allows quantitative analysis by optical density (OD) using a standard microplate reader. This convenient assay allows large scale screening and quantitative comparison of multiple samples and includes individual migration controls for each sample.
|Materials Required but Not Delivered||1. Precision pipettes: sufficient for aliquoting cells.
2. Harvesting buffer: EDTA or trypsin based cell detachment buffer, or other cell detachment formulations as optimized by individual investigators. Millipore’s ready-to-use non-mammalian detachment solution, Accutase™ (Cat. No. SCR005) can also be used.
3. Endothelial cells (for example: HUVEC cells).
4. Endothelium cell culture medium appropriate for subject cells, such as EGM-2 (Endothelial cell growth media-2)
5. Quenching Medium: serum-free medium, such as DMEM, MEM etc containing 5% BSA. Must contain divalent cations (Mg 2+, Ca2+) sufficient for quenching EDTA in harvesting buffer.
6. Sterile PBS or HBSS to wash cells.
7. Distilled water
8. (Optional) Chemoattractant or pharmacological agent added to culture medium.
9. Low speed centrifuge and tubes for cell harvesting.
10. CO2 incubator appropriate for subject cells.
11. Hemocytometer or other means of counting cells.
12. Trypan blue or equivalent viability stain.
13. Microplate reader (540-570 nm detection) or spectrophotometer.
14. Sterile cell culture hood
15. (Optional) Graduated ocular (calibrated), or automated method for counting stained cells on a membrane.
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Store kit materials at 2° to 8°C for up to their expiration date. Do not freeze.|
|Material Size||1 kit|
|Material Package||Sufficient for 12 assays|
|Reference overview||Pub Med ID|
|Positive influence of AP-2alpha transcription factor on cadherin gene expression and differentiation of the ocular surface.|
Judith A West-Mays, Jeremy M Sivak, Steve S Papagiotas, Jennifer Kim, Timothy Nottoli, Trevor Williams, M Elizabeth Fini
Differentiation; research in biological diversity 71 206-16 2003
The family of transcription factors Activating protein-2 (AP-2) are known to play important roles in numerous developmental events, including those associated with differentiation of stratified epithelia. However, to date, the influence of the AP-2 genes on endogenous gene expression in the stratified epithelia and how this affects differentiation has not been well defined. The following study examines the detailed expression of the AP-2alpha and AP-2beta proteins in the stratified epithelia of the ocular surface, including that in the cornea and developing eyelids. The effect of altered levels of the AP-2alpha gene on ocular surface differentiation was also examined using a corneal epithelial cell line and AP-2alpha chimeric mice. Immunolocalization studies revealed that, while AP-2beta was broadly expressed throughout all cell layers of the stratified corneal epithelium, AP-2alpha expression was confined to cell compartments more basally located. AP-2alpha was also highly expressed in the less differentiated cell layers of the eyelid epidermis. Overexpression of the AP-2alpha gene in the corneal cell line, SIRC, resulted in a dramatic change in cell phenotype including a clumping growth behavior that was distinct from the smooth monolayer of the parent cell line. Accompanying this change was an up-regulation in levels of the cell adhesion molecule, N-cadherin. Examination of the ocular surface of AP-2alpha chimeric mice, derived from a mixed population of AP-2alpha-/- and AP-2alpha+/+, revealed that a down-regulation in E-cadherin expression is correlated with location of the AP-2alpha-/- null cells. Together, these findings demonstrate that AP-2alpha participates in regulating differentiation of the ocular surface through induction in cadherin expression.Full Text Article
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|QCM™ 3 mm Endothelial Cell Migration Assay – Fibronectin, Colorimetric|