The MTT assay measures metabolic activity, which does correlate with cytotoxicity, but it does not specifically measure cytotoxicity. There are plenty of factors that can inhibit cell metabolism to some degree, without causing cytotoxicity. Typically there is also a concentration gradient, whereby increasing concentrations of a factor will inhibit cell metabolism, until at a certain concentration, that factor also induces cell death.

If you are just interested in cytotoxicity, then a better assay would be a live/dead dye viability assay. Cell undergoing necrosis or apoptosis almost universally feature plasma membrane permeability. If you have access to a flow cytometer, you can use fluorescent dyes such Propidium iodide, or TOPRO3 to assess live vs dead cell ratio. Dead cells will be brightly stained, live cells will not be stained. If you dont have a flow cytometer you can use a dye that is colorimetric, such as trypan blue, and then count blue stained cells vs non-stained cells in a Hemocytometer slide under a microscope.

The trypan blue assay is very rapid and inexpensive, but less accurate. The flow cytometer approach is more expensive, but also rapid, and more accurate/sensitive, but requires access to a flow cytometer.

Overall, to be sure of cytotoxicity you would ideally have untreated control cells (no alpha-syn) and test cells. Compare both against each other using the same assay. Further, you should test multiple parameters to establish cytotoxicity; in our lab we look at plasma membrane permeability with the afore mentioned dyes, assess cell metabolism with the Alamar Blue assay (similar to MTT assay), and also observe cell volume and size via coulter method (increasing electrical resistance due to larger cell volume; increasing cell volume can be an outcome of cell death processes), and morphology visually under microscope (in our case, as the cells die they detach from flask substrate, become more round, and there are more debris in the media).

We use mutiple parameters because sometimes cells become senescent, but dont die. Senescent cells will have similar morphological features (i.e. increased cell volume), and are metabolically less active, but do not have a permeable plasma membrane.

Gion Fränkl Thank you so much for the response. I did think of using MTT or LDH for measuring how A-syn fibrils cause protein aggregation in the N-27 Doperminergic cells and ultimately cause it to die. However, I wasn't sure how to correlate it or represent it as a result. I suppose MTT assay should work in my case or maybe I can just think about doing a Thioflavin T assay . Imaging should seems like a good option in this case.

Hi Sarah Voth Thank you so much for the suggestions. Yes, we do use N-27 cells lines which have DAT expressions and are sensitive to MPP+ neurotoxin. I have used these Neural cells before to assess potential therapeutic compounds for Parkinson's, so I believe they can be used to assess my amyloids. I feel hopeful and will surely test it out!! Thank you once again!