Hello Mariel Tavares Oliveira Prado Bergamo

It is of vital importance to obtain accurate, reliable results from the in vitro cytotoxic assays. A number of variables must be taken into account such as potential interferences, linearity, sensitivity and reproducibility of the assay. These assays must be sufficiently sensitive to detect small differences in cell number, yet robust enough to generate results that are reproducible.

Although the MTT assay results generally correlate with the number of viable cells growing in standard culture conditions, it is not so! The rate of tetrazolium reduction reflects the general metabolic activity. The rate of MTT reduction can also change with culture conditions (e.g., pH and glucose content of medium) and the physiological state of the cells. Moreover, MTT reagent exhibits cytotoxic effects and adding the reagent to estimate cell viability may actually be damaging or even killing cells during the incubation period.

Alamar Blue assay is more sensitive than tetrazolium assays. Also, it can be multiplexed with other methods to gather more information about the cytotoxicity mechanism. However, fluorescent interference from test compounds and the often-overlooked direct toxic effects on the cells are possible.

Other than Alamar Blue, I would suggest SRB assay could be a potential candidate to replace MTT assay. SRB assay measures total cellular protein content and does not rely on metabolic function or activity of live cells for quantification of cell number. This approach eliminates the influence of varying biological parameter like the metabolic rate or the cellular mitochondrial number on the quantification process.

Best.

I think there are some misunderstanding regarding this subject, I must first address those before I can answer your question:

First of all cytotoxicity is not the same thing as the viability. First one refers to the number of living cells and the second one represents a decrease in viability due to cell death. However since viability may decrease without cell death (ie: it may be caused by lower proliferation rate), they are not the same.

Secondly both tetrazolium assays (MTT, MTS, XTT, WST8 etc.) and Alamar Blue measures metabolic activity (via cellular dehydrogenase activity) and metabolic activity is not a very good indicator of cell viability as individual cells may have increased/decreased activity due to the treatment. Alamar Blue may have advantages over tetrazolium assays (such as increased sensitivity and specificity) but in the end both measure metabolic enzyme activity.

I would argue that the gold standard would be to count the number of viable cell via microscopy (you may have to use fluorescent dyes to differentiate living cells though). There are live/dead assays which uses a couple of fluorescent dyes to differentiate dead/dying cells from the viable ones.

SRB measures protein content which is assumed to be correlated with the number of living cells. There are other methods utilizing DNA content as an indicator as well. Such methods are somewhat raw (as the number of cells may be only partially correlated to the amount of total protein/DNA).

I recommend you to read our paper on this subject to learn more about such terms, common methods that are being used and how to provide evidence for different types of claims: Article From viability to cell death: Claims with insufficient evide...

Malcolm Nobre you provided good comments here and elsewhere. It bothers me that companies advertise viability and metabolic assays as "proliferation assays". I would ask though, because SRB measures protein concentration, would it ignore dying or apoptotic cells, as they have proteins that would be quantified?