Key Spec Table
|Description||QCM Laminin Migration Assay (24-well, colorimetric)|
|Overview||Also available: Cell Comb™ Scratch Assay! Get biochemical data from a scratch assay! Click Here
Cell migration is a fundamental function of normal cellular processes, including embryonic development, angiogenesis, wound healing, immune response, and inflammation (1, 2).
Cell migration may be evaluated using several different methods; the most widely accepted being the Boyden Chamber assay. The Boyden Chamber system uses a two-chamber plate model in which a porous membrane provides an interface between two chambers. Cells are seeded in the upper chamber and chemoattractants placed in the lower chamber. Cells in the upper chamber migrate toward the chemoattractants by passing through the porous membrane to the lower chamber. Migratory cells are stained and quantified. Most cells are sized from 30 to 50 µm can migrate through 3 to 10 µm pore.
|Materials Required but Not Delivered||1. Precision pipettes, sufficient for aliquoting appropriate volume of cells and reagents.
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) is recommended.
Note: Trypsin-based cell detachment buffer may be required for strongly adherent cell lines, but can strip cell surface proteins. Allow sufficient time for cell receptor recovery.
3. Tissue culture growth medium appropriate for subject cells.
4. Quenching Buffer: Serum-free medium such as DMEM or RPMI-1640, containing 5% BSA
Note: Quenching Buffer must contain sufficient divalent cations (Mg 2+ or Ca 2+) to quench any EDTA present in the Harvesting Buffer.
5. Chemoattractants (eg. 10% FBS) or pharmacological agents for addition to culture medium, if screening is desired.
6. Sterile PBS (Cat. No. BSS-1005-B) 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. Spectrophotometer (microplate reader with 540-570 nm detection capability).
13. Sterile cell culture hood.
14. (Optional) Graduated ocular (calibrated), or automated method for counting stained cells on a membrane.
|Safety Information according to GHS|
|Material Size||1 kit|
|Material Package||Sufficient for 24 samples|
|Reference overview||Pub Med ID|
|Wound healing--aiming for perfect skin regeneration.|
Science, 276: 75-81 (1997) 1997
The healing of an adult skin wound is a complex process requiring the collaborative efforts of many different tissues and cell lineages. The behavior of each of the contributing cell types during the phases of proliferation, migration, matrix synthesis, and contraction, as well as the growth factor and matrix signals present at a wound site, are now roughly understood. Details of how these signals control wound cell activities are beginning to emerge, and studies of healing in embryos have begun to show how the normal adult repair process might be readjusted to make it less like patching up and more like regeneration.
|Transient functional expression of alphaVbeta 3 on vascular cells during wound repair.|
Clark, R A, et al.
Am. J. Pathol., 148: 1407-21 (1996) 1996
During early granulation tissue formation of wound repair, new capillaries invade the fibrin clot, a process that undoubtedly requires an interaction of vascular cells with the wound provisional matrix composed mainly of fibrin, fibronectin, and vitronectin. Integrin alphaVbeta3 is the vascular cell receptor for these wound-associated adhesive proteins. Therefore, we investigated the expression of this receptor on new capillaries of healing full-thickness cutaneous porcine wounds. During granulation tissue formation, alphaVbeta3 was expressed specifically on capillary sprouts invading the central fibrin clot whereas the closely related integrin alphaVbeta5 failed to localize to these cells. Cyclic peptides or antibody antagonists of alphaVbeta3 specifically inhibited granulation tissue formation in a transient manner during the period of invasive angiogenesis. Immunolocalization studies revealed that alphaVbeta3 became aggregated and lost from sprouting vessels after treatment with a peptide antagonist. In contrast, beta 1 integrins were not modulated by this treatment. Once granulation tissue filled the wound and invasive angiogenesis terminated, the alphaVbeta3 showed little or no expression in the granulation tissue microvasculature. These data demonstrate that integrin alphaVbeta3 plays a fundamental, but transient, role during invasive angiogenesis and granulation tissue formation in a healing wound.
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|QCM™ Laminin Migration Assay|