MTT as I think

Dear Yibing Ruan,

that THE constant question. I regularly used different methods in my screening of small molecules and drug design or drug association. Crystal violet is only use in adhesion/desadhession cell assay. to have a high throughput screening, I used MTT assay. however that did not discriminate anti-proliferative effect, apoptosis, cell cycle nor metabolism alteration. This MTT/XTT assay is only based on NADPH enzyme activity of cells but may bring information of compounds activity on cells. The viability assay by high throughput screening depends if you have an access of flow cytometry that allows the used of 96-well plates to perform Annexin-V/propidium staining or fluorescent-caspase assays. In my lab, i d'ont have at this time thus i used MTT, then compound that have an effect on cell, are tested by facs using Annexin-V/propidium staining and cell cylce assay.

let me know

best regards

Hi Yibing,

as Yves already pointed out, it is good to not only look at only one assay system, since they mostly tell a different story.

I usually combine a viability assay with a cytotoxicity assay and if there are any indications an apoptosis assay might be included as well.

I use FDA (fluorescein diacetate) for viability, and LDH release assay for cytotoxicity since these can be easily combined from a single well. I use supernatant for LDH-release assay and cells for FDA afterwards.

If you need more details don't hesitate to contact me.

Best Kai

Thank y'all for the great input! I looked up info re FDA and it seems to be used for monitoring microbial enzymatic activity. How well does it go with mammalian cells? In particular, what's the linear range that corresponds with cell numbers? Thanks again!

Trypan Blue staining, MTT

Alamar Blue compared to MTT has different advantages including the possibility to read both fluorescence or absorbance, no cell lysis required and much higher sensitivity.

As clearly stated by Yves, "cytotoxicity" cannot be determined by means of the colorimetric MTT assay. You must use other tests than the MTT one if you want to measure cytotoxicity. The MTT test indeed enables you to evaluate "cell viability".

Once you have determined the GI50 / IC50 concentration for a given compound on a given cell line, you must indeed use complementary biochemical assays as recalled in mind by Yves, Kai and Giulo.

The two attached articles by Galluzzi and colleagues (2012, 2015; Appendix-1 and Appendix-2) are of great help.

The attached article by Kornienko et al. (2013; Appendix-3) reviewed various chemicals that are able to induce non-apoptotic cell deaths in cancer cells.

We think in our group that morphological techniques can also be helpful in addition to the MTT assay. Thus, coming back to the IC50 / GI50 values obtained by means of a colorimetric assay (as for example the MTT one):

* in the Mathieu et al. (2009 (Appendix-4) and 2015 (Appendix-5)) articles, the MTT test-related GI50 concentrations relate to actual cytotoxic effects.

* In the Lefranc – Nunzo et al. (2013; Appendix-6) 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.

* In the Van Goietsenoven et al. (2010; Appendix-7) 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; Appendix-6).

Lastly, you can also have "false" data generated with colorimetric assays (see the attached article by Chan et al. (2013; Appendix-8) and the NCI-60-cell line-related article (Shoemaker, 2006 (Appendix-9)).

The US NCI set up a fantastic tool to characterize the effects of a given drug in terms of growth inhibition in a panel of 60 cancer cell lines belonging to >10 histopathological types (Shoemaker, 2006; Appendix-9).

The US NCI clearly defined by means of the combination of the GI50 (growth inhibition), the LD50 (lethal dose by 50%) and the TGI (total growth inhibition) how to make the difference between a cytotoxic and a cytostatic compound:

The US NCI-related GI50 value corresponds to a global growth decrease by 50% induced by a compound on a given cell line “x” days after having cultured the cells with the drug and in comparison to an untreated control condition (= 100%) grown during the same time.

The US NCI-related LD50 value corresponds to the a global growth decrease by 50% induced by a compound on a given cell line “x” days after having cultured the cells with the drug and in comparison to the initial number of cells in the untreated control condition.

The TGI is the US NCI-related parameter to determine the concentration needed to kill 100% of the treated cells.

It is by comparing the GI50 to the LD50 value that one can determine whether a compound is cytotoxic or cytostatic, and not at all with the sole GI50 value.

We are using morphological approaches in the research unit to which I belong for determining whether a compound is cytotoxic or cytostatic (see Lefranc-Nunzo et al., 2013 (Appendix-6); Mathieu et al. 2009 (Appendix-4), 2015 (Appendix-5); Van Goeitsenoven et al., 2010 (Appendix-7)).

The US NCI is not so far for having tested about 800,000 anticancer drugs, whose data are publicly available on the NCI website https://dtp.cancer.gov/databases_tools/data_search.htm

I actually benefited several times from the amazing help of the NCI in identifying the mechanism of action of an innovative anticancer compound (see for example Frederick et al. JMC 2011 (Appendix-10)).

Best regards

Robert

Are you using proliferating cultures and treatment time of more than ~4 h? Then please read this. Viability assessment with proliferating cultures requires single cell analysis and cannot be done in a 96-plate format using a plate reader. I wrote the following section for the most misleading crystal violet "cytotoxicity" assay which applies to all 96-well plate "viability" assays. A major advance in cancer research will be made when such misinforming and useless assays will be removed. "The Crystal Violet "Cytotoxicity" assay with proliferating cultures will determine anything but cytotoxicity. The 2016 protocol description by Feokistova et al. is for non-proliferating cells. Please see their Fig. 1 (control wells before and after treatment; there is no change in cell number!!!). In fact, the description of all such 96-well plate assays (MTT, CellTitre-Glo, etc) for viability assessment with proliferating cultures are majorly flawed. Misinformation perpetuated by misuse of such assays, as well as large-dye uptake assays (trypan blue, PI) have been discussed (PMID 30071623 and refs therein, including PMID: 27750219). If you use the single-cell MTT assay described in PMID 30071623, you will see that cells that are assumed to be dead (even with apoptotic morphology) are viable and metabolize MTT. Like the "gold standard" colony formation assay, all 96-well plate "viability" or "cytotoxicity" assays that are performed with proliferating cultures determine the ability of a test agent to convert proliferating (dangerous) cancer cells to dormant (most dangerous, tumor repopulating) cancer cells, and not to dead cancer cells. The IC50 values measured by such assays reflects a sum of cell cycle checkpoint activation (a pro-survival mechanism), short-term dormancy due to reversible apoptosis (anastasis), and early stages of durable proliferation arrest (dormancy, due to polyploidy, multinucleation and senescence, all of which contribute to disease relapse). None of these well-documented responses is associated with loss of viability. Viability assessment with proliferating cultures requires single cell assays and cannot be determined in a multiwell plate format using a plate reader."