Q: What endothelial cells are compatible with this assay?
A: We use HUVEC cells, but any endothelial cells can be used.
Q: How can I confirm that the insert is evenly covered with endothelial cells?
A: After culturing the endothelial cells on the insert for 2-3 days, the insert can be stained with Crystal Violet solution (0.1% in 10% ethanol) and viewed under the microscope. Usually 50K to 100K healthy endothelial cells will be sufficient to form monolayer after 3 days.
Q: How do I stimulate cells with TNF alpha?
A: We recommend treating endothelial cells with 20ng/ml TNFalpha between 4 to 12 hours.
Q: Do the inserts need to be used all at once?
A: The inserts provided with our Transmigration assays are individual and removable inserts that do not need to be used all at once. Any unused inserts can be stored at 4ºC until needed and can be use with any 24-well sterile tissue culture plate.
Q: Can I use cells that express GFP with this assay?
A: The Transmigration assays 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 the LeukoTracker and CytoTracker dyes, despite having the same emission wavelength.
Q: How can I improve the results of my transmigration assay?
A: In general, with migration assays we recommend using the highest cell number with the lowest possible migration time. It is recommended to use an incubation time at the low end of the range (2-4 hours) to reduce the chance of any random migration events that tend to happen with longer incubations, which can skew the results. The optimal incubation time will depend on the cells being used and should be optimized. We recommend using the maximum amount of cells (300,000 cells) to get the highest level of detection, because with even the most highly migratory cell line, a maximum of just 5% is expected to migrate.