effect of substance on viral infection - (Nov/22/2013 )
Am designing an experiment to check the effect of a substance on the viral infection using a cell line grown in 96-well plate. The main aim is to see if the infection rate increases or decreases at a single concentration of the substance. Now there are two ways to design this experiment.
1) you seed cells in 96-well plate and dilute down the virus starting at 1:50 dilution all the way to 1:32000 along with a starting concentration of the substance. This essentially means the substance will get diluted but will be equally diluted along with the virus.
2) you seed cells in 96-well plate and dilute down the virus starting at 1:50 dilution all the way to 1:32000. And then add an equal concentration i.g: 10ug/ul of the substance through out the dilutions. This would essentially mean that 100 virus or 10 virus (as you go down the dilution) will be subjected to 10ug/ul.
Which one is correct, I would think both needs to be done to eliminate the drawbacks but there is very little of the substance.
The analysis of viral infection will be carried out using flow cytometry
Actually, your second option is viable, given that you want a standard concentration of the drug.
There is a third way - seed cells, add an equal amount of virus to each well, dose with varying amounts of the treatment... That's the option I would go for, but doesn't work if you want to keep the level of drug constant, but it is a better measure of the biological activity of the drug.
Hello thank you for your reply.
But don't you think it would not be logical to subject low virus numbers to the same concentration of the substance. The high concentration would invariably effect the infectivity of the virus??
The third option suggested seems a good one except I have to use a constant concentration of the substance.
Tests of biological activity should cover a range where it does have an affect, but also where there is no affect. Changing the concentration of the drug and the virus at the same time is madness - how do you measure the output in a case like that? You need to look at one variable at a time.
Suggest you determine infective dose is for the virus in this assay and hold that steady vs. differing concentration of substance. Have you any concept as to mechanism? If directly upon the virus, you might combine substance and virus before exposure, if on cell so as to make cell less vulnerable - you might add substance and them virus, if on cell such that viral reproduction following penetration is blocked - might add it with or after the virus.