I performed MTT assay for assessing the cytotoxicity of curcumin (0-50uM) on LPS-stimulated THP-1 macrophages, and used DMSO as control vehicle. While calculating the cell viability% for DMSO, I found that cell viability was more than 100%. What could be the possible reasons for this result?
Hi,
viabilities above 100 % are not unusual, especially in the MTT assay. This can have two main reasons, one is random experimental fluctuation (should be within +/- 10 %), the other is stimulation by the treatment ('hormesis'). MTT is prone to compounds interferring with energy metabolism (e.g. uncouplers), which can increase MTT metabolism to up to 200 % baseline activity. However, don't interpret this as "the cells are doing better than without the compound". In the MTT assay, you (overly simplified) measure cellular respiration, and its induction can indicate severe cellular stress.
For DMSO, I did not see any effects on the cells yet, but it is very well possible that your cells respond to the solvent. However, for your experiment, this should not be a problem if you adjust the DMSO concentration in all concentrations of your compound to the same levels.
What values above 100 % are we talking about here exactly? A third possibility is direct chemical reduction of MTT by your compound, however when I observed this, values were excessively high (>500%).
Proliferation? Is it possible that there was proliferation? I see it in my doctorate when I did MTT assay to evaluate cell viability from NRK52E cells , a type of adherent cells. I did 3 measures, one control at 0 hour of exposition to Fe3+, one after 3 hours of exposition to Fe3+, and a third after 24 hours of exposition, between control and 3 hours, there was difference only between the groups that received Fe3+, where these groups showed a decreased in viability, what it was expected, but there was a tendency in control group to be higher at 3 hours compared with 0h control. But when I analyzed the 24 hours group, the difference it was really different, there was an increase in cell viability. When I did immunofluorescence marked for ki-67 the results suggested that for this kind of cells, 21 hours it was sufficient to complete the cell cycle (data not published), and increase the proliferation in these cells. When I searched information about the duration of the cell cycle for this kind of cells, I found one work of 1976 showed that the duration of cell cycle for this kind of cells it was around 19 to 21 hours. But ki-67 this is not the better marker for see the proliferation, because it only mark one early phase of the proliferation. To see proliferation the better marker it is PCNA. Another method which you can use to see cellular viability, better than MTT assay it is the bromo uridine incorporation method. This because the non-specificity of MTT assay. There are some works from 2013 showing that the formazan crystals do not are formed only in mitochondria but in cytoplasm also, and because of it this is not a so reliable assay to test cellular viability, even that, some works was published at Scientific reports in 2017 using this technique (MTT assay) to determine cellular viability, and I used this technique to test cellular viability also in 2018, I have just published my work using this technique. You can find my work in my RG profile. The figure that showed the cellular viability in my case it was the figure 6.
I hope that it helps you.
Pedro
Hi Pedro
Thanks for the reply. The cells I used were THP-1, already differentiated into macrophages by PMA. So, there cannot be any proliferation.
Maybe, contamination? Did you repeated this experiment and reach the same result?
How are you diluting the curcumin? Curcumin it is a salt with an intense yellow color? Maybe the way that you are diluting it is causing some precipitation or because the yellow color of curcumin it cause some diffraction in the length of the laser that you are using to read it. What it is the wave length which you are using? 500 nm?
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