Most of the papers on antiviral studies use MTT assay to check the cytotoxic concentration of any new molecule (synthetic or natural). This assay gives an idea about the maximum non-toxic dose of the compound. My question to the experts is that is MTT assay the right choice and will it be reproducible in two different lab conditions given that the compound and the cell lines are the same.
Also, I would like to get some light on what is the range of non-toxic concentrations that should be used for antiviral testing as we know that In a cell culture model, apparent antiviral activity of an investigational product can be the result of host cell death after exposure to the product.
MTT, MTS, Resazurin etc. all give a good 'quick look' at viability and are cheap and easy to use. As long as variables are controlled there should be good reproducibility lab-to-lab. It can also be used as add-mix-read multiple times as long as you change media between reads - although care should be taken to avoid contamination.
However, they all rely on the reduction of the reagent by mitochondrial enzymes, so if your drug inhibits mitochondrial function/induces metabolic shift you may get a mis-leading results from these assays, so keep in mind where is might not be appropriate to use for all drugs.
Alternate end-point cell death assays include CellTox, Calcein AM/PI or trypan blue exclusion assay which will tell you if cells are dead or dying, and are good for follow up studies.
As for concentration, you will have to run some validation assays as this is likely to change compound to compound, but you can check for cell death using the assays above.
Good luck.
Hi Piya,
I would suggest for SRB based cytotoxicty assay, as it measures total cell mass, without any biochemical interference. Measure both cell proliferation (cell growth) and cell viability to find out GI50, TGI, and IC50; this will reveal both cytostatic and cytotoxic concentrations.
What is the best antiviral screening assay in cell culture?
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