Hello, Good day Innocent Agida

The optimal MOI is known for many cell lines. If you are unable to locate the known MOI for your cells, you need to perform a pilot experiment determining the MOI by first transducing your cells with lentivirus expressing a fluorescent reporter such as GFP or mCherry.

Follow these steps:

1. You may select a few MOI’s to test. For instance, using a GFP Lentivirus, design a range of MOIs to use, let’s say you may start with a minimum MOI of 1 and then proceed with 5, 10, 15, and a maximum of 30. It is always better to test a lower MOI range to avoid cytotoxicity at the higher MOI’s. If you have any reference suggesting the appropriate MOI for your cells, then you could devise the range around the suggested MOI.

2. After you have infected your cells, allow the appropriate time to pass before evaluating the fluorescence. For lentiviruses, this is generally 48 -72 hours post infection. Then record the fluorescence at the various MOI's to determine your transduced cell percentage.

3. You may select the minimum MOI at which all the cells (100% infection) are expressing the transgene.

Then you may follow the protocol provided in the link below.

https://www.genecopoeia.com/wp-content/uploads/2018/03/Lentivirus-protocol-GeneCopoeia.pdf

Some calculations you would want to know.

For instance, if you need an MOI of 10 and if your virus titer is 1 x 10^6 IU/ml and you would like to deliver to 1 x 10^5 cells, what volume of the virus would you need?

Use the formula,

MOI=Virus Titer × Virus Volume/Total Cell Number

10 = 1×10^6 IU/ml × X / 1×10^5 cells

X = 1ml

So, you will need 1ml of virus to achieve an MOI of 10.

Hope this helps!

Best.

Thanks very much for your response, I appreciate! Malcolm Nobre