Cultrex Collagen I Cell Invasion Assay

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.

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.

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.

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.

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.

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 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).

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.

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.