Q: Can the plates in your Anoikis Assays be re-used?
A: Our Anoikis assay uses a hydrogel coated plated that cells cannot attach to, which promotes anoikis cell death. The hydrogel integrity on the surface of the plate may not be maintained throughout the assay protocol and we don’t recommend re-using the wells. The plates used for the Anoikis assay can be used over multiple experiments and do not need to be used all at once. If you plan to use the plate over multiple experiments, the plate can be stored covered at 4C and we recommend that you sterilize the unused wells by exposing the plate to UV light under a cell culture hood for 30 minutes before use.
Q: Can I save the unused wells to use later?
A: The 24-well plates used for the Anoikis assay do not need to be used all at once. If you plan to use the plate over multiple experiments, the plate can be stored covered at 4C and we recommend that you sterilize the unused wells by exposing the plate to UV light under a cell culture hood for 30 minutes before use.
Q: How does MTT and Calcein AM work?
A: MTT is used for proliferation assays and measures the activity of enzymes in living cells that reduce tetrazolium dye (yellow) to formzan (purple). Calcein AM is also used as a live cell indicator because it is cleaved by intracellular esterases once it enters the cells, which converts calcein to a green fluorescence. Either MTT or calcein AM can be used to measure cell viability, depending on the type of plate reader available.
Q: Will this kit differentiate cells that have died by anoikis rather than apoptosis?
A: Anoikis is a form of apoptosis that is induced by the absence of proper cell attachment to the extracellular matrix. Our Anoikis assay uses EthD-1 dye to measure cell death, which is not specific to anoikis or apoptosis. However, the assay uses a hydrogel coated plated that cells cannot attach to, which promotes anoikis cell death. Although the assay does not differentiate between apoptosis and anoikis, cell death with this assay can be assumed to be from anoikis based on the assay conditions.
Q: The provided plate is clear. How do I take a fluorescence reading for the Calcein AM stain?
A: Our Anoikis assays come with a clear plate to which cells cannot attach, and this promotes anoikis cell death. The cells can be transferred to a black walled plate and read in a fluorescent plate reader for Calcein AM detection.