Q: How is chemotaxis measured using a Boyden Chamber system?
A: When using a Boyden Chamber, the cells are in serum-free medium in the upper chamber and the media with the chemoattractant is in the lower chamber, which establishes a chemoattractant concentration gradient between the two chambers. This gradient is maintained through surface tension, which prevents the chemoattractant from readily diffusing into the upper chamber. Migratory cells are able to reorganize their cytoskeleton when in a chemoattractant gradient in order to move toward to the higher concentration area.
Q: How is the chemoattractant gradient maintained between chambers during the assay?
A: Surface tension between the upper and lower chambers prevents the chemoattractant from diffusing to the upper chamber.
A: If you are in the early stages of your migration 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: When using your Chemotaxis Assays, how can I guarantee that during the incubation with Cell Detachment Solution any non-migratory 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: Why are the cells resuspended in serum free media?
A: It is recommended to resuspend the cells in serum free media in order to establish the chemoattractant gradient. Serum contains many cytokines and growth factors and any serum present will act as a strong chemoattractant and mask the affect of the chemoattractant being tested.
Q: Is it necessary to culture cells in serum free media before using migration assay?
A: Starvation is not required for all cell migrations. Some researchers starve their cells (usually cancer cell lines) because starved cells usually respond well to chemoattractant treatment.
Q: What controls should I use when measuring migration towards a chemoattractant?
A: Our recommendation is to use controls consisting of cells in serum free media with migration towards: 1) serum free media, 2) 10% serum media, and 3) serum free media with the chemoattractant. It is important that there is no serum in the media with chemoattractant you are using because any serum present will act as a strong chemoattractant and mask the affect of the chemoattractant you are testing.
Q: What pore size should I use for my cell line?
A: Please see our chemotaxis assay selection by cell type guide on our website: http://www.cellbiolabs.com/chemotaxis-assays. A literature search can be performed to determine Boyden chamber pore sizes successfully used by other researchers for specific cell lines that are not listed here.
Q: Do I need to use the entire plate at once, or can I save unused wells for future tests?
A: For 24-well migration assays, the inserts are individual and removable units and any unused inserts can be stored at 4C for future use. The inserts can be used with any standard sterile 24-well plate. To use the 96-well plate over multiple experiments, it is important that care is taken not to spill any solutions into the unused wells. Store the plate at 4deg or RT, then before using for the second experiment expose the plate to UV light in a cell culture hood for 10-20 minutes.
Q: Can I use GFP expressing cells?
A: Either the colorimetric or fluorometric kit can be used with GFP expressing cells. The GFP signal is expected to be low and will not contribute much to the overall signal of CyQuant fluorescence used in the fluorometric kits, despite having the same emission wavelength.
Q: Can I go longer than the incubation time specified in the manual for my treatment?
A: The migration assays were designed to study chemotaxis of cells within a few hours and we don’t recommend an extended incubation period, which can increase random migration events.
Q: Do you offer a colorimetric version of the 96-well assays?
A: Unfortunately we are not able to offer a 96-well colorimetric migration assay because the detection is not sensitive enough to detect the small number of cells expected to migrate with this assay.
Q: How can I improve the results from my chemotaxis assay?
A: Our recommendation to get the highest level of detection is to seed with the maximum number of cells in serum free media and incubate for a short amount of time (4-6 hours). Using the maximum cell number is important because even with the most highly migratory cell line under ideal conditions, a maximum of just 5% to 10% are expected to migrate. Running a cell dose curve will confirm if there are enough cells above the background level of detection. It is also important that the cell detachment and cell lysis steps are performed thoroughly and consistently.
We recommend running the following controls with our Cell Migration Assays, which consist of cells in serum free media with migration towards:
1. Serum free media
2. 10% serum media
3. Serum free media with the desired chemoattractant at various concentrations.
Q: Can suspension cells be used with migration assays?
A: Suspension cells can be used with our migration assays containing 3 µm and 5 µm pore sizes. Cells that are not strongly adherent will form two populations during the migration assay: cells that attach to the membrane and cells that fall through the membrane. These migration assays have a slightly different protocol that is designed to detect cells that fall through to the bottom of the well in addition to cells that attach to the bottom of the insert.
FAQs for Specific Chemotaxis Assays: