Dear Paul,

Mixing lentivirus and adding them directly onto the cells may be an option ; nevertheless I would highly recommend you to use Magnetofection method for this specific application.

In brief the Magnetofection is a quite recent way of transfecting and transducing/infecting cells by associting magnetic nanoparticles with biomolecule-to-deliver (DNA, siRNA, mRNA, lentivirus, adenovirus....). Under the attraction of a specific magnetic field, magnetic vectors are concentrated onto the cell surface within minutes and the transfection/transduction and infection are enhanced.

This method was extensively used in many papers with lentiviruses and I would highly recommend Sacha JB's articles that used ViroMag nanoparticles to enhance infection of CD4+ T cells with HIV. Moreover, they demonstrated that this is the only method allowing synchronizing infection (Sacha JB., Nat Protoc. 2010 Feb;5(2):239-46 ; http://www.ncbi.nlm.nih.gov/pubmed/20134424).

This last point, is to my point of view very important regarding your experiment. Actually lentivial transduction is seen by cells as a viral attack and innate mechanisms are activated to lower the infection or to alert surrounding cells that virus are coming (INF, IL paracrine secretion...). If you add all your viral particles directly onto cells, as soon as the first one is detected, infection of the other will be hampered.

Using Magnetofection will allow you improving your experiment by :

- lowering the MOI for an enhanced efficiency and a lower toxicity

- complexing the 3 viral vector on the same magnetic nanoparticle (as soon as the cargo enters, the 3 virus also enter)

- and as explain above, synchronizing the infection: as all the virus are concentrated withtin minutes onto the cell surface, the signal will not have the time to alert other cells.

I will be very pleased to give you more information on this method that I'm sure will be a valuable asset for your experiment.

would you have any question please do not hesitate to contact me directly at : [email protected]

good luck with your research,

best regards,

Cedric