Hello Kathi Ging

The optimal polybrene concentration will vary depending on the cell type used. Polybrene can be toxic to certain cell types for instance, primary neurons. For human fibroblasts, you may use polybrene with a working concentration of 5ug/mL. You could also perform cytotoxicity assay to evaluate the toxic effect of polybrene on your cells.

You may change to fresh media 24 hours post-transduction. However, if viral toxicity is observed in your cell line, you may decrease the infection time to between 4 to 20 hours by removing the virus-containing media and replacing with fresh media.

Best.

I used to perform transduction of marrow cells with lentiviruses using 4 ug/ml of protamine sulphate and used to change my media on the same day post transduction. Polybrene in general is toxic to most of the cells and can be used at a concentration of 8 ug/ml and you can change the media post transduction. I used to do dendritic cells transduction with LVs in presence of polybrene (8 ug/ml) and used to change my media on the same day and it has worked quite nicely.

Thank you for your suggetions. Polybrene 8 ug/ml with changing medium after 24 hours worked nicely without causing toxicity in this fibroblass line . Transduction efficency was greatly enhanced.

Hi Kathi,

We are also trying to transduce human fibroblasts with lentiviral vector. We can get good efficiency with empty AcGFP and mCherry vectors but not much efficiency when we have an insert in there using spinfection and 4ug/ml polybrene overnight. Just wondering what seeding density your are using for the fibroblasts. We have about 80-90 confluency in our in 6 well plates.

kind regards

Steve.

I am seeding them at a much lower density (40-50% confluency at time of transduction). I infect them in 12 well plates to save some virus and later split them into larger vessels (6 well plates). Hope this helps.