Since you are using MOIs of 5 and 10, you must know the titer of the virus. What method did you use to determine the virus titer?

Hi Tom ,

Thanks for getting back to me , I used the Takara Bio lentiviral titration p24 Elisa kit and after obtaining the standard curve , I calculated the TU/ml .

Thank you for the clarification.

I don't think there is anything wrong with your transduction process. The problem most likely the titer of the virus. It is falsely high due to using p24 to quantify. Using p24 tells you the total amount of virus present but does not tell you what the infectious titer is, which is what you really need to know in order to achieve the desired transduction efficiency. In any virus population you will have virus particles that are able to infect and particles that are defective in some way (missing the RNA genome, are unable to adsorb/infect, etc). If your construct has a fluorescent marker (GFP) you can easily determine the infectious titer. If not, you will have to increase the MOI based on your p24 calculated titer until you reach your desired transduction efficiency.

Good Luck!

Tom Masi : Thank you so much for the explanation . This has been really helpful .