I never used the SHSY-5Y cell line.

However, I can tell you that you cannot determine "cytotoxicity" by means of a colorimetric 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.

When you obtain 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 (Alley et al., 1988 (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)).

Hoping that this long explanation would not be too boring,

Best regards

Robert

Dear Prof. Robert,

Thank you for the detailed reply, It helps me to understand the logic of cytotoxicity.

My aim in present experiment was just to find out LD50 of LPS on cell line of my interest, as few papers suggested LD50 concentration of LPS can be used to produce ROS. so as to induce oxidative stress using appropriate concentration of LPS.

I am basically working on neurological disorders so at this point I need not to go for Apoptosis and other assay for toxicity.

My aim was just to produce ROS which is not working with LPS. so what may be the problem in your opinion whether my cells are not responsive to LPS or some other issues? 

Dear Shubham,

Thank your for your reponse.

Unfortunately, I am not at all an expert in neurological disorders and I do not want to speculate on an answer.

I let RG specialists in this domain coming back to you.

Best regards

Robert

Thank you Professor Robert Kiss

Dear Shubham

 do you have references in literature that the concentrations you have tested have some toxic effects on these cell lines? Maybe the concentrations are too low to induce effects on cell proliferation and MTT, that "count" viable cells", answer correctly underlining no effects. Are the cells less than control if you observe them on a microscope? You can try to "kill" cells using TWEEN or SDS as positive controls.

Yes indeed Diego!

Robert

Perhaps the number  of cells per well is too low

Dear Diego Baderna, 

Thank you for the response

I do not find any clear protocol for the concentration of LPS to be used with SHSY-5Y cells. My aim here is not to kill cells. I am trying to optimize protocol for ROS estimation using LPS as inducing agent, where I need a concentration which do not kill cells but is able to produce significant ROS. So MTT is just a preliminary study to finalize the concentration of LPS to be used. That is the reason I used 7 different concentration of LPS. Moreover I doubt whether MTT is suitable for these cells or not ? Also I doubt the no. of cells is probably less, I am thinking to try with 20000 cells/well.

Please suggest me if something else can be done

Dear Asha Khanna, 

I also feel that no. of cells is less.

Any suggestion regarding density of cells to be used?