Q: What is the detection limit with the CyQuant® dye?
A: The limit of detection with CyQuant® is about 500-1000 cells, which is less sensitive than manual counting. Our recommendation to get the highest level of detection is to seed with the maximum number of cells (500,000) in serum free media, because with even the most highly migratory cell line under ideal conditions, a maximum of just 5-10% are expected to migrate.
Q: What is the minimum number of cells that can be added to the insert?
A: The minimum amount of cells that should be added to the migration assay is 10,000 (0.1x10^6 cells/ml) but the optimal cell number will vary based on how migratory the cell line is. Our recommendation to get the highest level of detection is to seed with the maximum number of cells (500,000) in serum free media, because with even the most highly migratory cell line under ideal conditions, a maximum of just 5-10% are expected to migrate.
Q: Will non-migratory cells that are attached to the upper insert be detached with the detachment solution? Can I scrape these cells from the top of the insert?
A: Following migration, migratory cells are attached to the underside of the polycarbonate membrane and are detached after incubation in cell detachment solution. Any non-migratory cells remaining in the upper chamber will not go through the membrane during the detachment. A scraping step is not included with the 96-well migration assay because it is too difficult to swab inserts in the small wells of the 96-well plate.
Q: Is the detachment solution harmful to cells?
A: The Cell Detachment solution is not harmful to the cells and allows for the cells to be recovered and re-cultured or used in downstream applications. If you do not plan on recovering the cells, the effects of the detachment solution will not be a concern because the cells are lysed following detachment. The cell detachment solution typically works within 10-20 minutes depending on the cell line, although the solution is gentle enough for cells to be exposed for extended time periods.
Q: Will the color of the media interfere with the reading?
A: The pink/red color from phenol red in the cell culture medium has no effect on the fluorescence reading of CyQuant dye used in these cell migration and invasion assay.