Cultrex Collagen I Cell Invasion Assay
Cultrex Collagen I Cell Invasion Assay SummaryThe Cultrex Collagen I Cell Invasion Assay provides a tool for assessing cell invasion through Collagen I.
Why Use Cultrex Collagen I Cell Invasion Assay?
Since different cell lines and different treatments can result in a wide range of invasive potentials, the permissiveness of each cell line to invade through Collagen I may be optimized to fit each experiment by adjusting the coating concentration. The Cultrex Collagen I Cell Invasion Assays are provided as either a single 96 well plate providing capacity for large screening experiments or as 24 individual inserts providing flexibility for smaller studies.
For research use only. Not for diagnostic use.
FBS Stimulates Migration of NIH-3T3 and HT1080 Cells. The NIH-3T3 mouse embryonic fibroblast cell line and the HT1080 human fibrosarcoma cell line were treated with 10% fetal bovine serum (FBS). The migration of untreated (yellow bars) and treated (green bars) NIH-3T3 and HT1080 cells against different extracellular matrix components, including Cultrex BME, Laminin I, Collagen I, Collagen IV, were quantified using the Cultrex Cell Invasion Assay Kits (Catalog # 3455-096-K, 3456-096-K, 3457-096-K, 3458-096-K, respectively). Data from four experiments was quantified for both non-invasive (NIH-3T3) and invasive (HT1080) cell types.
Citations for Cultrex Collagen I Cell Invasion Assay
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Angiopoietin-like Protein 2 Is a Multistep Regulator of Inflammatory Neovascularization in a Murine Model of Age-related Macular Degeneration.
Authors: Hirasawa M, Takubo K, Osada H, Miyake S, Toda E, Endo M, Umezawa K, Tsubota K, Oike Y, Ozawa Y
J Biol Chem, 2016-02-02;291(14):7373-85. 2016-02-02
A novel molecular pathway for Snail-dependent, SPARC-mediated invasion in non-small cell lung cancer pathogenesis.
Authors: Grant J, Fishbein M, Hong L, Krysan K, Minna J, Shay J, Walser T, Dubinett S
Cancer Prev Res (Phila), 2013-11-19;7(1):150-60. 2013-11-19
What is cell invasion?
Cell invasion is cell migration through a physiological barrier in response to a chemoattractant, and this recapitulates cell movement within a physiological environment which is composed of extracellular matrix proteins. Cultrex® Cell Invasion Assays evaluate cell invasion based on the cells ability to traverse membranes that are coated with a layer of extracellular matrix proteins. The cells must traverse this barrier through a combination of protein degradation and cellular locomotion.
What are the variables associated with cell invasion?
The major variables associated with cell invasion are cell type, cell density, composition of the physiological barrier, thickness of the physiological barrier, chemoattractant that is used, and time of culture.
Will the Cultrex® Cell Invasion Assay work for my cells?
The Cutltrex Cell Invasion Assay is currently configured for invasive adherent cell lines. If your cell line is adherent and there is evidence in the scientific literature that your cell line is invasive in a Boyden chamber, it should be compatible with our assay. If this is unknown, then the invasive potential will need to be determined empirically.
How should cells be cultured prior to setting up the Cultrex Cell Invasion Assay?
Cells need to be healthy and actively dividing in 2-D culture. Cells should be passaged two or three times after resuspension from cryopreservation, and they should never surpass 80% confluency during each passage. Cells should also be assessed for viability using trypan blue, and they should exhibit less than 5% staining.
How do the Cultrex Cell Invasion Assays compare to wound healing assays?
Wound healing assays, also known as scratch assays, monitor cell migration laterally on a tissue culture treated plate. This is accomplished by generating a void in a cell monolayer by either removing cells or treating the surface of the plate to prevent cell growth in a designated area. The assay measures the ability of the cell monolayer to fill this void, and it may be conducted in the presence of extracellular matrix proteins. Since this assay is conducted within one chamber, there is no chemotactic gradient, and without the membrane, the cells are no longer required to change shape and squeeze through the pores. Another potential problem is that this assay does not control for differences in cell proliferation. While wound healing assays may be valuable for supplementing the Boyden chamber assay, it is not a replacement.
Can the Calcein-labeled invasive cells be subcultured?
Calcein AM cytotoxicity should be determined empirically for each cell line or model. For best results, the cells should be removed from the cell dissociation solution and placed in fresh culture medium as soon as possible.
The kit comes with both black and clear microplates. Do I need to use both?
The entire assay may be conducted in either the clear or black assay plate (provided in3455-096-01), if desired. The black assay plate provides increased sensitivity and reduced background for plate readers that read from the top. The clear assay plate provides compatibility for plate readers that read from the bottom. Some researchers have used the clear assay plate for the invasion step and the black assay plate for detection (top read).
In the Cell Invasion Assay protocol, how would you recommend aspirating off the coating solution from the top chamber as described in step 7?
This is best done manually with a pipette, not a vacuum. The collagen gel must remain in the well, therefore care should be taken to avoid disturbing the gel during aspiration.
Will the gel be removed when aspirating the top layer?
No, the gel must remain in the well. Refer to step 7 of the Cell Invasion Assay protocol - only the solution will be removed. It is best to manually remove the solution with a pipette instead of using a vacuum.
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