I have to perform permeability assay on 6.5mm diameter cell culture inserts with MOI 10. The virus titre is 10^6 pfu/ml. The required cell count per insert is around 10^5 cells/ insert. So, the bottleneck is the MOI I plan to infect and the volume of the virus needed to attain it as the area of the culture insert can allow 350ul maximum for addition.
MOI of 10 is very high, if you cannot reduce your MOI, you will have to concentrate your virus since you are limited on volume, .35 of your virus can only get you 3.5 MOI with that cell number. Without knowing what virus I cannot tell you for certain, but using PEG to precipitate it and reducing volume of resuspension can increase titer by 10-100 fold of most viruses.
Alternatively, if there is no other way, then you could try using 12 wells, instead of 6.5mm inserts. or if you have to use the 6.5mm dish only, then follow as Annette said. or otherwise, try to get higher virus titer, by reinfecting the indicator cells and concentrate the virus.
Hi Annette,
I am using dengue virus for my infection studies and I tried to passage it more but surprisingly, the titre started dropping after Passage 12.
You can use an amicon filter to concentrate your virus 3X. It should not take more than an hour, i guess.
Hi Sneha Singh,
If I understood correctly your problem is: after pas.12 (every with MOI 10) the virus titre fall down. MOI 10 - is very high. As a result you have a lot of defect virus particles in virus population after pas.12 and the virus titre must be not so high.
Thank you for your suggestion. The question that I am going to ask is about dose dependent modulation of the vascular leakage. So, I would like to perform the experiment for proper understanding of the process.
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