Hi Tushar,

I think that in the method I, at the final stage you are further diluting the virus 15 folds (20 uL topped up with 300 uL). I would have calculated and prepared a working dilution containing 80 000 virions in 300 uL of fresh media (x the total volume required for all wells) so that 300 uL can be added to the wells (NO topping up with media, hence no further dilution). This is what your method 2 says. I just have one suggestion- You can first dilute your stock to 10 folds (directly add 45 uL media/ diluent to the 5 uL of your stock eppendorf tube ), so that the amount of stock taken for further dilution can be increased to 17.68 uL. A voulme of 1.768 is very much likely to cause pipetting error and hence altered MOI.

Let me know if it works,

best wishes,

SD

Sibnarayan Datta Thank you for your response. To my surprise, I actually saw my cells today and the infection seemed to have worked even after adding the 280 uL to the 20 uL of virus.

Regardless, I agree with the fact that method 2 is better and that it should be diluted by 10 fold to reduce error.

We actually have stocks of 5 uL in each eppendorf, and since HSV-2 does not remain as efficient after it is freeze-thawed, we usually throw away the left over virus from the 5 uL. So, I was thinking why not use all 5 uL? Moreover, this would also reduce error even more.Adding 5uL of virus in 33825.8 uL media and then taking 300 uL of that diluted virus should also work, right?

Thanks for your help.

Dear Tushar,

I agree with you. Its always better to use the stocks once they are thawed.

I hope you'll get expected results this time.

best wishes,

SD

" don't I still have 80 000 virions in each well regardless? So the MOI is still 1" Yes your are right the ratio number of virus/number of cells is still 1. The volume of infection is not (very) important; it should cover the cells to avoid them to dry. (of course if you put viruses in a very large volume there is little chance that they could contact the cell) . remember that with a moi of 1 only 2third of the cells will get at least one virus (so 1/3 will not be infected at the first round of infection)(Poisoon's law)

Didier Poncet Thank you for your input. I was thinking the same thing, which is why I topped it up with the 280 uL to avoid dryness. Moreover, now that I think about it, both methods are correct, because essentially, you end up with 266 virions/ uL considering that I'm adding 80 000 virions in each well with 300 uL media in total for each well.

Maybe next time I'll cut the volume down to make sure there is higher chance of virus and cell contact.

Thank you.