Q: Is the colorimetric or fluorometric kit more sensitive?
A: The fluorometric assay is more sensitive than the colorimetric assay, however manual counting can be performed with the colorimetric assay following the staining step and prior to the extraction step. Manual counting is always more sensitive than any detection method, including fluorometric.
Q: How does the stain in the colorimetric assay work?
A: Our invasion assays use a staining solution that will fix and stain cells regardless if they are alive or dead. The stain will not be washed away with water but will instantly be dissolved in the kit extraction solution. The extraction solution is then read at 560nm in an ELISA reader.
Q: Is the invasion chamber plate in a strip well format?
A: The ECM Invasion Chamber Plate is a 24-well plate containing 12 individual inserts. These inserts are removable and any unused inserts can be stored at 4ºC until needed.
Q: Is there another cell line that can be used as a positive control?
A: MDA-231 can be used as an invasive positive control if HT-1080 is not available.
Q: How can non-specific stain on the insert be removed?
A: After staining the inserts, the non-specific stain can be cleaned with a q-tip, which will make it easier to see the remaining stain that was missed the first time. It may help to wet the q-tip with water and press firmly against the bench to flatten the end. This will allow the q-tip to get into the corners of the insert. If the extraction has already been performed, the insert can still be re-stained because any invaded cells will be attached to the bottom of the insert. If the problems removing the stain are not resolved, the cells can always be counted manually.
Q: Can the inserts be re-used for subsequent experiments?
A: The inserts in our 24-well Cell Invasion Assays cannot be re-used because the insert is coated with basement membrane proteins, which will no longer be intact after running an experiment. The kits are provided with 12 individual inserts so it is possible to store any unused inserts at 4ºC until needed.
Q: Is it normal for invasive cells to show a patchy distribution after invasion?
A: Yes, this is a perfect result. For migration assays, most cells will be evenly distributed after staining. The invasion assay is different, since the cells must penetrate through the ECM layer. There will be some very aggressive cells that digest this layer and make a hole, which allows other cells to follow through.
Q: Can cell viability be measured with a hemocytometer after invasion?
A: Cells are still viable following the cell detachment step and an aliquot of the cells in the cell detachment buffer can be used to measure cell viability. Although the cells are still viable at this step, it may be difficult to measure viability because of the low cell concentration of invaded cells and the low sensitivity of the hemocytometer. One cell visible in the hemocytometer is equivalent to 10,000cells/ml. With migration and invasion assays, only 5-10% of cells are expected to migrate for even the most aggressive cell line. If you use the maximum amount of cells in the assay, 300,000 cells, and 10% were to migrate, you would still only have 30,000 cells per assay.
Q: My cells require a different media. Is this OK?
A: Before running the assay, a cell suspension is prepared in serum-free medium. The cells do not need to be cultured in this media.