The tested drug is a chemotherapeutic agent
the type of the cell lines is colorectal cancer cell lines
the cell viability was compared to the control
my thought was the cells were activated by these small concentrations
Can the experts give me a scientific explanation about this situation, please?
And why is happened?
Indeed, depending on the dose, some compounds can switch from enhancing proliferation to being cytotoxic. However, you should check your assay meticulously to discard simpler technical explanations. For example, you can dedicate cell-free wells to blank with vehicles and treatment.
Dear Shiekhah!
This situation can still be when you have more cytotoxicity (cell death) in control than in experiment. For example, with the same MTT in the control wells, the optical density may be lower than in the experimental wells. This may be due to both the type of cells, the type of analysis, and the time of exposure to the chemotherapy drug. For example, some cells (fibroblasts) metabolize MTT for a long time (4 hours), cancer cells (glioma) - faster (2-2.5 hours).
In the MTT test, sediment or poor solvent can also be the cause, when the pharmaceutical grains in the wells do not completely dissolve. For example, when I did MTT, DMSO did not dissolve crystals well and I got such results. I started using isopropanol with 0.04 N HCI
Alexandr Chernov
regarding to my question, I always use Isopropyl as solvent to dissolve the mtt crystals
and the control wells are healthy there are no apparent dead cells either in the wells or from the culture flask
is this situation referred to the hydrophobic Property of the drug?
Do You have more cells? Are they dividing more after small amount of compound tested?
It is not unusual that small amounts of a toxic substance or even the solvent produce an initial increase in viability. You may want to read about the concept of hormesis:Article Hormesis Defined
In the case of the MTT assay, mitohormesis might play a role because the outcome depends on mitochondrial dehydrogenase activity (https://www.nature.com/articles/nm.3624).
There are many ways to assay cell viability.
Which method, precisely, did you use? All are indirect, with the exception of counting cells directly (not using a machine).
Your results will also be influenced by the carrier/excipient you used to deliver your pharmacological agent, the presence of serum, the density of your cells, how 'old' they are (how long in culture), when you last passaged them, accuracy of the T°C & CO2 regulators in your incubator, age and storage conditions of your media; so many variables!
To provide an intelligent answer, I need more details!
Adam Figarski
Hi Adam,
yes, as I mentioned in the question
i have more cells (the cells divided more than the control wells ) when they exposed to the smaller concentrations of the tested drug.
Carsten Lange
Thanks carsten for defining this situation and many thanks for the article that is a helpful and interesting one.
David Ian Leavesley
Hello David,
i used mtt assay which is a mitochondrial dependent assay.
Of course David on the next days, I will use the counting with Trypan blue dye as I used in my master thesis and I will compare the mtt results to the the counting ones to determine which method is more sensitive and accurate.
thanks for your suggestion.
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