Hi!
We are producing lentivirus for T cell transduction. However, there are several protocols that do not mention the use of polybrene for titration in HEK293T, but they use centrifugation instead. My question is, is polybrene necessary? Our virus expresses GFP, so we can titrate using flow cytometry.
Thanks for helping!
Hi Gonzalo,
Polybrene is used as a transduction enhancer. I don't think is that important with HEK293T cells because they are adherent which already improves transduction. Moreover, if you use polybrene in titre plates it could mask the real titre by enhancing transduction, but I don't think that matters too much.
Also, regardless of whether you use polybrene or not you will still need to use a flow cytometer for GFP-based titration.
When performing your T-cell transduction you should then use polybrene or other enhancing reagents. In my lab, we prefer RetroNectin from Takara Bio, especially when transducing suspension cells like T cells, since it mediates the physical union between lentiviruses and cells, dramatically enhancing transduction.
Good luck!
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