I'm testing 3 different incubation times: 24h, 48h and 72h. For 24h incubation, viability starts at 100% and maintain 100% even in higher concentrations, which means it has no toxicity effect on my cell.
For 48h incubation, viability starts and maintains at 60%. And, at 72% incubation starts at 20% and with the increasing of concentration the viability increases until 100% too. Can someone explain me these results?
Thank you in advance.
I must admit that I do not understand very well your question because it seems to me that in your question text you mixed several time "viability %" and "hours".
If you are speaking about the effects of a given compound at a given concentration on cell viability, yes indeed the time of cell incubation in presence of the drug will markedly influence the data.
Usually, at a given concentration of a given drug on a given cell line, the viability of these cells will decrease with increasing incubation times of the cells with the drug.
A great majority of the established cancer cell lines routinely used in the labs over the World display doubling time rangins between 18 and 25h.
Thus, for certain cancer cell lines a period of incubation of 24h with a given drug is too short to obtain "significant" activity on cell viability.
The US NCI has tested (and continues to actively test) not far from one million compounds.
They analyzed the drug-induced effects on cancer cell population growth after 48h.
We use 72h of cell-drug incubation time in our research unit.
However, unfortunately, cancers in patients are very heterogeneous and their cell doubling times amazingly changes from one subclone to another one in the same cancer.
We have to keep in mind that some "poor cancer cells prinsoners of plastic flasks" are far to be representative of the "biological reality" in a cancer developing in a patient.
Thus, I think that it is better to "stay" on 48 or 72h to be able to compare "our" data to those generated by our colleagues.
I am attaching here articles relating to the NCI 60-cell-line approach and also some articles on the complex biology in "true" cancers.
Best regards
Robert
I agree with Robert. I would also make sure you are seeding an appropriate number for cells per well. I have had problems in the past, especially with fast growing cell lines, where my control cells would over grow and this stress would skew the viability comparisons.
Hi Carolina,
If you are using an MTT/MTS assay to measure cell viability, I'd recommend a few things:
1) First, prepare an assay where you plate different number of cells and measure the viability over time (without any drugs first). The number of live and metabolically active cells will be directly proportional to the absorbance you get. As Richard mentioned above, you do not want cells to reach 100% cell confluency before the end of your experiment. Depending on the cell line you're using, cells will get stressed, die and detach from the plate... messing up your results.
2) After you determine the appropriate number of cells for 24, 48 and 72 hours (let's say you choose to use a cell number that gives you 50% of your saturation absorbance), then you can add different drug concentrations raging from 100% viability (lower concentration) to 0% viability (highest concentration). Of course, that may require you to do a couple of experiments to test different concentrations before setting up your final experiment.
I have used this kit in the past: https://www.promega.com/~/media/files/resources/protocols/technical%20bulletins/0/celltiter%2096%20aqueous%20one%20solution%20cell%20proliferation%20assay%20system%20protocol.pdf
They have some good tips on assay optimization too.
Hope this helps. Best of luck!
Jackie
Hi Robert Kiss, I have a question regarding the incubation time with MTS .
Usually it is mentioned that incubation for 1-4 hours with MTS reagent before reading. I have tried cancer cell line(incubated for72 hours with drug) for 1 hour, 2 hour, and 3 hour incubation with MTS reagent. The viability % is different according to time. So, which data should I believe on..1 hr,2 hr or 3 hr?
How to determine the MTS reagent incubation time for different cell lines?
Thank You
Kamal Pandey
Dear Kamal,
I never used MTS, but only MTT.
If you want the precise details about how using the MTT test, please contact my colleague Prof. Véronique Mathieu ([email protected]) on my behalf.
Best regards
Robert
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