If you just want to replace the dye, there are many alternative, for instance XTT or WST1 (which is easy-to-use and, therefore, recommended by me).

If you want to address another cellular indication system besides mitochondrial dehydrogenases you could use, for example, an assay aiming at ATP. Promega offers some easy-to-use assays.

My preference would be to use tritiated thymidine.  Much more sensitive relative to MTT. 

Cell counts and fluorescent viability dyes are another approach.  I am guessing you don't really mean anti-proliferative but rather cytotoxicity / apoptosis.  Lots of ways to semi-automate this. 

clonogenic assay on plastic or in soft agar is a less high throughput but very reliable.

For adherent cells, xCELLigence is the way to go. It is the most sensitive method, and you get proliferation data in real time, not end point. Also, you just plate cells and walk away, no other hands on procedure. 

The best way is to assay both cytotoxicity and viability. For cytotoxicity, measure of LDH release is a straightforward fluorescent assay, amenable to medium throughput: it measures the amount of dead cells after a chronic or acute insult. There several kits available in the market that make your life easier, but it is also simple to make the assay reagents in house. For viability, I would choose ATP production. There are also several kits in the market easy to optimize, such as ATPlite.

I prefer MTT followed by DAPI assay would give you broad view of drug action on the cell.

Nice response from Marta!

And now, my boring and so repetitive comments as I already made a lot of time on RG about "MTT and cytotoxicity".

A colorimetric assay cannot assess "cytotoxicity".

Cytotoxicity means "cell killing effects".

A colorimetric assay can only bring "growth inhibition information" because it is a relative test in which you compare the ODs of a treated condition to the ODs of a control condition arbitrarily scaled at 100%.

When you obtain a concentration (for a given compound) decreasing by 50% the global growth (after x days), you do not know whether you killed 50% of the cells (cytotoxic effects), whether you inhibit 50% of the cell proliferation (cytostatic effects), whether you detach 50% of the cells (anti-adhesive, i.e. "in vitro antimetastatic" effects), etc..., etc...

Once you have determined the IC50 concentration for a given compound on a given cell line, you must use complementary biochemical and/or morphological techniques to determine whether your compound is cytotoxic, cytostatic, anti-adhesive, etc..., etc...

Some of the attached articles could also be of help.

For example, in the Mathieu et al. (2009 and 2015) articles, the MTT test-related GI50 concentrations relate to actual cytotoxic effects.

In the Lefranc – Nunzo et al. (2013) article, the MTT test-related GI50 concentrations relate to cytotoxic effects that in turn do not relate to apoptosis … This means that each cytotoxic effect does not “universally” translate into pro-apoptotic ones (see the Kornienko et al. (2013) article).

In the Van Goietsenoven et al. (2010) article, the MTT test-related GI50 concentrations relate to cytostatic effects, neither to cytotoxic nor to pro-apoptotic ones.

Be aware that you cannot always translate the MTT test-related growth inhibition of a given compound into a precise GI50 value. Some compounds reach a “plateau” of inhibition (see Lefranc – Nunzo et al., 2013).

Lastly, you can also have "false" data generated with the MTT colorimetric assay (see the Chan et al. (2013) attached article).

The attached articles by Galluzzi and colleagues (2009, 2012, 2015) perfectly illustrate how mixing various tests to be able to actually “speak” about cytotoxicity.

Best regards

Robert

There are many approaches for evaluation of cellular proliferation but at different endpoints, and provided below are five examples. The best assay depends on the purpose of your study.

[1] A simplest way is to directly count cell numbers at different days after plating.

[2] A colorimetric assay based on MTT, XTT, MTS, WST-1 or WST-1 measures the enzymatic activity of mitochondrial dehydrogenase (succinate-tetrazolium reductase) that converts these tetrazolium salts into purple colored insoluble formazan. A similar assay employing resazurin or AlamarBlue can be used both colorimetrically and fluorometrically. The assay of this type evaluates the mitochondrial metabolism.

[3] Tritiated thymidine uses radioisotope and evaluates the DNA synthesis. A similar assay employing other thymidine analogues BrdU, IdU, CldU or EdU does not need the use of radioisotope. There have been S phase markers available, such as Ki-67, PCNA, DNA topoisomerase II, and histone H3. Evaluation of E2F (required for a transition from G1 to S) or other proliferation-related markers may also be useful.

[4] A dye exclusion assay measures the membrane permeability and uses colorimetric (e.g., trypan blue, Erythrosin B) or fluorescent dyes (e.g., propidium iodide, ethydium bromide).

[5] A colony formation or clonogenic assay measures the ability of single cells to form colonies with 50 or more cells, usually in about two weeks after plating.

The above is a good list.  I believe the thymidine is the most sensitive.  Note the exclusion exclusion assays are viability.  Badu and case can also be used.

Many thanks, Prof. Talmadge.

The reason why I included the dye exclusion assay in the list is that cell numbers can be counted simultaneously along with evaluation of viability.

A point sometimes misunderstood is that a colony formation or clonogenic assay does not evaluate viability.

On one hand, resulting colonies with 50 or more cells are considered as survivors (surviving clonogenic colonies), based on which the surviving fraction is determined. On the other, colonies with less than 50 cells are called abortive colonies.

Cells composing clonogenic colonies are not necessarily viable, and cells composing abortive colonies are not necessarily non-viable.

Very painful to count a number of samples.  You had a good list.