A: If you are in the early stages of your invasion studies and are still optimizing conditions, it is recommended that you start with the 24-well format. You can move to the 96-well format after conditions are optimized and you feel comfortable with the assay protocol.
Q: What is the difference between your product #CBA-101-COL where collagen is pre-coated on the underside (bottom) of the membrane, and your product #CBA 111-COL where collagen is coated on top of the membrane?
A: These products assay for different processes – cell migration and cell invasion, respectively. We offer four different types of cell migration assays, and you can get more information on the various definitions here. Our assay #CBA-101-COL is designed to measure haptotaxis, where immobilized ECM proteins act as the chemoattractant toward which cells migrate. Our assay #CBA-111-COL is designed to measure cell invasion, which is a more complex process that involves cell migration, degradation of the ECM, proteolysis, and movement of cells through the ECM to neighboring tissues. In this assay cells must be able to express active proteinases such as MMPs in order to digest and move through (i.e. invade) the coating on the top of the membrane.
Q: When using your Cell Invasion Assays, how can I guarantee that during the incubation with Cell Detachment Solution any non-invasive cells remaining in the top chamber will not pass through the membrane to the chamber below?
A: The membrane pore size used in a Boyden chamber assay should always be smaller than the diameter of the cell to prevent passive movement of cells through the pores. The cell must rearrange its actin/myosin cytoskeleton structure in order to extend its leading edge and allow it to pass through the pores of the membrane. The 30 minute incubation with Cell Detachment Solution does not allow enough time for cells to do this.
Q: Do you have colorimetric invasion assays in a 96-well format?
A: We offer colorimetric cell invasion assays only in a 24-well plate format. Unfortunately the colorimetric detection is not sensitive enough for these assays to be provided in 96-well plates.
Q: How do I select between basement membrane and collagen?
A: Most cell invasion studies will use the basement membrane coating on the inserts. The collagen gel coated inserts are only for studies that are interested in invasion mediated by collagen.
Q: Why is serum free media used on the top and 10% serum on the bottom?
A: Serum is commonly used as a chemoattractant for cell migration and invasion assays because serum contains many cytokines and growth factors. By resuspending cells in serum-free media, a chemoattractant gradient is established between the cells in the upper chamber and the 10% serum in the lower chamber, which gives the cells an incentive to invade through the gel layer.
Q: Is it necessary to culture cells in serum free media before the assay?
A: Some researchers starve their cells (usually cancer cell lines) because serum contains many cytokines and growth factors and starved cells usually respond well upon chemoattractant treatment. It is not necessary to perform an overnight starvation; instead you can resuspend your cells in serum free media prior to starting the assay.
Q: What are the components of the ECM?
A: The inserts of our basement membrane invasion assays are coated with an ECM gel that is the same as Matrigel. The ECM extract was prepared from Engelbreth-Holm-Swarm (EHS) sarcoma produced in mice and contains laminin as a major component as well as collage type IV, heparan sulfate proteoglycan, entactin, and other minor components.
FAQs for Specific Cell Invasion Assay Kits: