Normally MTT assay is used for evaluating the anticancer activity of synthesize compounds. How much MTT is reliable?
The MTT assay measures the ability of cells to reduce the tetrazolium dye, MTT, to its insoluble formazan, resulting in a purple color. Since this requires a functioning mitochondria, the MTT effectively measures the metabolic activity of live cells. It is quick, cheap, easy to perform and relatively reliable. This is why it is used for drug screening.
However, there are some caveats. First, you must ensure that you are measuring activity at the most active part of the cells growth phase, which means you should do a growth curve optimization for your cells prior to doing any drug screening. Also, this is an absorbance-based assay which means it is not very sensitive at picking out minor changes. And, most importantly, it measures metabolic activity, not necessarily viability. Some cells can be perfectly viable but not necessarily with a lot of metabolic activity. So it is always important to validate your results using something that directly measures cell apoptosis and/or necrosis.
The MTT assay measures the ability of cells to reduce the tetrazolium dye, MTT, to its insoluble formazan, resulting in a purple color. Since this requires a functioning mitochondria, the MTT effectively measures the metabolic activity of live cells. It is quick, cheap, easy to perform and relatively reliable. This is why it is used for drug screening.
However, there are some caveats. First, you must ensure that you are measuring activity at the most active part of the cells growth phase, which means you should do a growth curve optimization for your cells prior to doing any drug screening. Also, this is an absorbance-based assay which means it is not very sensitive at picking out minor changes. And, most importantly, it measures metabolic activity, not necessarily viability. Some cells can be perfectly viable but not necessarily with a lot of metabolic activity. So it is always important to validate your results using something that directly measures cell apoptosis and/or necrosis.
As Marianna said, the MTT assay is easy and inexpensive enough that it could be used as a preliminary method for screening. It should *NOT* be used as primary evidence for cytotoxicity or anti-proliferative activity because it measures mitochondria as an indirect measure of cell count.
Some compounds such as epigallocatechin gallate (EGCG) promote mitochondrial function and biogenesis, which causes artificially high MTT readings due to the increased amount of mitochondrial activity. This could nullify possible decreases in cell count or cause a compound to be mistaken as pro-proliferative.
(see P. Wang "Limitations of MTT and MTS-Based Assays for Measurement of Antiproliferative Activity of Green Tea Polyphenols", PLoS ONE)
Although MTT is not a perfect assay for cytotoxicity, but it is quick and cheap, and it's good for initial screening. My experience is, double check with your eyes under the mic (before add the MTT dye) and to see whether the cell numbers are in concordance with your MTT results. You can perform further cytotoxicity/cell proliferation/in vivo assays to verify interesting findings. For effective anti-cancer drugs, their effects must be obvious enough for us to see by bare eyes. Otherwise, I doubt the whether it is a good drug to killl the tumor cells. All the best!
You may refer to the following paper for reference (MTT was initially used to screen for 20 diffrent anti-cancer reagents although it is not published in that paper).
Cheung AK, Ip JC, Lung HL, Wu JZ, Tsao SW, Lung ML. (2013) Polo-like Kinase Inhibitor Ro5203280 Has Potent Antitumor Activity in Nasopharyngeal Carcinoma. Mol Cancer Ther.12(8):1393-401.
MTT is one of simple, inexpensive and reliable test to screen the cytotoxicity of a drug. But for drug with an action on mitochondria it will be not reliable and u should carry another kind of cytotoxic assays.
please also check the ideas and suggestions of this topic
https://www.researchgate.net/post/Can_a_potent_apoptotic_drug_interfere_with_MTT_assay_for_cytotoxicity
According our laboratory experience in cytotoxicity assay, I'd like to inform you that MTT on the one hand is quite reliable, but there are many limitations. We use it in correlation with SRB and Crystal violet staining. Only with one assay you may have ''positive false'' data. Check this article for advantages/disadvantages of each stain.
https://www.researchgate.net/publication/235993742_1._AnvirzelTM_in_combination_with_cisplatin_in_breast_colon_lung_prostate_melanoma_and_pancreatic_cancer_cell_lines?ev=prf_pub
Article 1. AnvirzelTM in combination with cisplatin in breast, colon...
I have noticed some limitations when using the MTT assay for cells cultured under hypoxia. I mean, you cannot compare normoxic cells vs hypoxic cells, their metabolic status is different.
So what would you all recommend me to use when i have two option PrestoBlue and MTT.
I am testing the cytotoxicity of my compound on Hela cell line.
Thank you!
The current debate is so interesting that I would also like to obtain some additional and CHALLENGING references from you.
I am responding to you in two separate sections.
The current one describes my very personal view about "MTT versus cytotoxicity".
In the second one, I will send you some additional references about "problems" that can be encountered with colorimetric assays in general, and with the MTT one in particular.
Best regards
Robert
"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 enables you to evaluate "cell viability".
Cytotoxic means direct "cell killing effects" induced by the drug of interest.
Cytostatic means that the compound of interested lowers the growth rate of a given cell population without direct cell killing effects. Cell death will occur as a consequence of a too long cytostatic effect.
A colorimetric assay can only bring "relative global growth inhibition information" because it is a relative test in which you compare the ODs of a treated cell population to the ODs of a control condition (untreated cells) arbitrarily scaled at 100%.
Thus, the IC50 / GI50-related values obtained by means of a colorimetric assay do actually not translate “cytotoxic” effects.
If one wants to determine actual cytotoxic effects for a compound of interest, the MTT colorimetric assay can be completed with the lactate dehydrogenase (LDH) test.
LDH is a soluble cytosolic enzyme that is released into surrounding culture medium upon cell damage or lysis during apoptosis or necrosis for example. The quantitative determination of LDH in the cell culture medium can be used as a marker for cytotoxicity.
Coming back to a colorimetric assay such as the MTT one, when one obtains a concentration (for a given compound) decreasing by 50% the global growth (after x days (usually 2 or 3)), i.e. the GI50 concentration (or the IC50 as commonly used in the literature) you do not know whether your compound of interest killed 50% of the cells (cytotoxic effects), whether it inhibited 50% of the cell proliferation (cytostatic effects), whether it detached 50% of the cells (anti-adhesive, i.e. "in vitro antimetastatic" effects), etc..., etc...
Once you have determined the GI50 / 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...
The two attached articles by Galluzzi and colleagues (2012, 2015; Appendix-1 and Appendix-2) are of great help in this domain.
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.
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 first 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)).
Since some days i have argued with my assistant about the MTT assay.
Thanks Robert for your comprehensive answer.
In MTT, If the % Cell Viability is 70% Whether we can say it shows cytotoxicity
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