Some comments about this Q&A exercice.

The IC50 / GI50-related values obtained by means of a colorimetric assay do actually not translate “cytotoxic” effects.

Cytotoxicity means "cell killing effects".

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%.

When you obtain a concentration (for a given compound) decreasing by 50% the global growth (after x days), 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) are of great help in this domain.

The attached article by Kornienko et al. (2013) 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 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.

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 attached article by Chan et al. (2013) and the first NCI-60-cell line-related article).

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 (Alley et al., 1988).

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 my research unit to determine whether a compound is cytotoxic or cytostatic (see Lefranc-Nunzo et al., 2013; Mathieu et al. 2009, 2015; Van Goeitsenoven et al., 2010).

The US NCI is not so far for having tested about one million of 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).

Hoping that this long explanation would not be too boring,

Best regards

Robert

I was wondering how do you calculate your LDH assay data? If you simply calculate % LDH released in medium, you should consider how much LDH activity your cell type releases to medium "at baseline" in resting/untreated state. I work with various macrophage models and I´ve noticed that untreated macrophages do release some 5-10% of their cellular LDH-activity in culture medium over time. This baseline value does not reflect the actual cytotoxicity induced by your treatment, so you should subtract this backround to avoid overestimation of the cytotoxic effects of your treatment (the LDH Kit should provide a simple formula to do this).

I remove the baseline and also the LDH values are represented as %negative controls, so that the estimation is corrected. 

Be aware that your LDH results can be interfered by many reagents! Especially bivalent cations like zinc or calcium ions can greatly influence your LDH analysis.

These two assays evaluate 2 different phenomena. You can compare the results, but try discussing these values considering that cytotoxicty from MTT is a different phenomenon from the membrane viability analyzed by LDH assay (since MTT assay measures the mitochondrial activity). It has happened to me too.

I hope it helps!

I agree with Kristiina, the establishment of a "baseline" is essential, but you also have to determine the amount of the LDH activity maximally releasable by your cells (100 % or positive control) for a signal range calibration. This is usually achieved by lysis using detergents like Triton X-100, the 0 % baseline is obtained from a simple negative or solvent control, respectively.

Remember that the signal in the LDH assay is inverse (higher signal means lower viability) to what you usually observe in other colorigenic assays like MTT or NRU (where higher signal means higher viability).

If you use serum supplementation, be aware that a significant amount of LDH activity may be accounted to the serum, so make a blank measurement of culture medium without cells, too.

Also remember that MTT and LDH are totally different endpoints, as Luis mentioned. MTT reducing activity can be found in lysates, too, so cells with damaged membranes may not react in the MTT assay, but in the LDH. Vice versa, toxicants may disrupt cellular respiration, but leave membranes intact, therefore only showing toxicity in the MTT assay. Try to interpret the results on basis of the toxicity meachanisms your compounds are known for.

http://www.ncbi.nlm.nih.gov/pubmed/1793087

http://www.ncbi.nlm.nih.gov/pubmed/21053782

Measurement of cell viability and proliferation forms the basis for numerous

in vitro assays of a cell population’s response to external factors. The reduction

of tetrazolium salts is now widely accepted as a reliable way to examine

cell proliferation. The yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2,

5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part

by the action of dehydrogenase enzymes, to generate reducing equivalents

such as NADH and NADPH. The resulting intracellular purple formazan can be

solubilized and quantified by spectrophotometric means.

The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely,

when metabolic events lead to apoptosis or necrosis, the reduction in

cell viability. The number of assay steps has been minimized as much as possible

to expedite sample processing. The MTT Reagent yields low background

absorbance values in the absence of cells. For each cell type the linear relationship

between cell number and signal produced is established, thus allowing an

accurate quantification of changes in the rate of cell proliferation.

I suggest:

1. subtraction of background

2. considering the probable interferences of the toxicant that you are determining its effects on the cells

3.considering MTT and LDH are totally different endpoints, as Luis and patrick mentioned, try to interpret your findings on the basis of the assessment mechanisms of two methods and the used toxicants

Hi Mubin,

I agree with Luis Patrick and Ameneh, LDH and MTT indicate different cytotoxicity endpoint. I suggest using a known positive control as I think that your tested toxicants are not known. After that you can compare obtained results from both LDH and MTT.

Regards

Nina

THANK YOU SO MUCH 

Hello. I have the exact same problem for one of the tested materials. The LDH release assay indicates that the material is toxic (200% of LDH release, compared to the control which is cell culture medium, assumed as 100%) but the MTT results indicate the opposite (90% of cell viability). Did you find an explanation for your results? Did you find any paper reporting the same effect MTT vs LDH?

Hi,

remember that the effect on the signal in the LDH assay is inverse to what you usually observe. A low signal means a low activity of LDH in extracellular medium and therefore high cellular viability. Inverse treatment-signal-relationships are exactly what you expect when directly comparing the results from LDH assay with e.g. MTT assay, where low signals mean low viability.

Don't use your negative control measurements as 100 % viability baseline as you would do in MTT assays, but acutually calculate cell mortality with your positive control signal as 100 % reference (e.g. by addition of detergents).

Without intention for advertising, maybe take a look at the second graph on this website, and things may become clearer: https://www.thermofisher.com/order/catalog/product/88953

Thank you Patrick, I know that.

My positive control for cell death (lysis buffer) presents a LDH release of around 500% (comparing with the control, assumed as 100%). So, I know that the cell viability control with the LDH release assay is around 60%, but with the MTT assay is around 90%. I just want to understand why, but cannot find any paper with similar results. With other tested materials, the results from LDH release and MTT assay correlate.

I have a theory that the material acts as a metabolic enhancer and is internalized by the cells. Thus although with a lower number of available cells (as indicated by the LDH assay), MTT conversion into formazan is accelerated, resulting in the overestimation of the cell viability. However it’s just me trying to explain this!

But I really need to find papers where this occurs in order to seek an answer.

Hi,

as you described, you calculate your effects with your negative control signals set to 100 % viability, however, you do not assess viability in LDH as you would in the MTT assay, but you measure cytotoxicity or cell mortality, respectively. In LDH, the actual negative control signals are not (!) what you refer to, but the positive control signals, which represent 100 % mortality by definition. Actually, you would have to set your negative control signals to 0 % cytotoxicity for correct calculation, so they technically respresent something similar to a blank measurement (activity present in medium with fully viable cells).

Again, your positive control signal cannot ever become 500 %, because it is set to 100 % mortality by definition. Right now, you are making a mistake by setting your negative control signals to 100 %, which is right for most assays (e.g. MTT), but not for LDH.

Refer to the figure posted above and compare the two test methods LDH and almar Blue. As the signal increases for LDH with increasing treatment concentrations, almar Blue signals decrease almost symmetrically. From what you explain, it sounds to me that you observe something similar in those two tests (just swap almar Blue for your MTT assay here), which is however something you would actually expect given from the different nature of both approaches.

To make things more clear, try to plot both results into the same graph similar to the graph on the website (or individual graphs) and post them here, it is always easier to discuss such matters on actual data.

Some comments about this Q&A exercice.

The IC50 / GI50-related values obtained by means of a colorimetric assay do actually not translate “cytotoxic” effects.

Cytotoxicity means "cell killing effects".

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%.

When you obtain a concentration (for a given compound) decreasing by 50% the global growth (after x days), 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) are of great help in this domain.

The attached article by Kornienko et al. (2013) 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 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.

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 attached article by Chan et al. (2013) and the first NCI-60-cell line-related article).

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 (Alley et al., 1988).

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 my research unit to determine whether a compound is cytotoxic or cytostatic (see Lefranc-Nunzo et al., 2013; Mathieu et al. 2009, 2015; Van Goeitsenoven et al., 2010).

The US NCI is not so far for having tested about one million of 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).

Hoping that this long explanation would not be too boring,

Best regards

Robert

Hi,

In our studies toxicity evaluations were performed using two cytotoxicity assays instead of just one. This is because certain chemicals have been reported to give divergent results in different toxicity tests including the NRU and MTT assays (Olivier et al 1995; Chiba et al 1998). Besides, Evans et al (2001) have recently found that in some cases one of the NRU or MTT assays can be more sensitive in detecting the toxicity of non-viral transfection reagents.

*****

Evans, A.R., Alexander, D., Burke, P. and Reed, C.J. (2001) Toxicity and transfection efficiency: comparison between four commercially available non-viral transfection reagents in a human bronchial epithelial cell line. British Pharmaceutical Conference Science Proceedings 2001,  106.

******* 

Evans, A. (2003) In Vitro and Ex Vivo Studies on the Toxicity and Efficacy of a Selection of Non-Viral Transfection Reagents.  PhD Thesis, Liverpool John Moores University, Liverpool, UK.

*********

Please also see attached paper. 

I am having a similar case with you. And the explanations do not seem to be relevant to the point in question.

Yes, you can get cytotoxicity values with MTT (inversely) and when you compare with cytotoxicity obtained from LDH assay the two are completely different. The results (MTT and LDH cytotoxicity values) on the same experiment were widely different.

I don't know if anyone has an explanation?

Find one possible explanation here: http://www.sciencedirect.com/science/article/pii/S0141813016324060

Regards

I think it is a good and relevant explanation in your paper @ Luis Braz. The same observation may be applicable to our case (the compound and assays).

Thanks