I am in the act of performing an MTT assay to check cell proliferation and cytotoxicity. I am confused about choosing a lysing buffer to dissolve the salts. Papers suggested:
1. NP40 with 4mM HCl and isopropanol;
2.4N HCl in isopropanol; and
3. DMSO.
Can anybody help in choosing the better one from the above three solutions?
Hi, I've just saw this paper that you might be interested to look into while preparing your protocol. You can download the pdf through this link:
http://link.springer.com/article/10.1007%2FBF02439354?LI=true
All the best!
I apologize for those couragous followers of mine ... and I know that I am bothering many of them by repeating so often the following few items.
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
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