Hi,

Depends on the cell type, and benefits are usually seen with optimized transduction parameters. We use it regularly at 8ug/mL on mouse BMMCs.

Goodluck!

Hi Murtaza,

I need to transduce fetal liver HSCs. Thanks for your response!

If the envelope is VSVG, several positively-charged polycations such as polybrene, DEAE-dextran, protamine sulfate, poly-L-lysine, or cationic liposomes will increase the transduction. However the concentration for each reagent should be optimized for primary mouse cells.

Naina,

Why do you want to do with only protamine sulfate? polybrene 8 ug/ml worked well for me with jurkat cells. It works well.

Or you can try spinoculation method for infection to Hsc cells.

Or, Use high MOI.

Or take 50000 cells in 100 uL of media containing virus+polybrene or any reagent. Incubate this mixture in Falcon in incubator overnight. Next day transfer to 24 well plate. I have tried this and it worked.

Hope these ideas may help you, Good luck.

Hello Ran Zhou & Mohammad Irfan,

Thank you both for suggesting the different possible ways of doing this.

@Mohammad- I have tried all of these already but the results are not very exciting, which is why i want to explore other agents such as protamine sulfate. I have had great success with using polybrene to transduce cell lines. But as i mentioned, I'm trying to transduce primary cells (murine fetal liver HSCs) which has been a bit of a challenge so far. I have tried transducing these cells by spinfecting with high viral MOI in presence of different concentrations of polybrene (2-8ug/ml), but not only the transduction eff. is low but the cells look unhealthy in polybrene.

I'm currently trying different conc. of protamine sulfate with spinfection method, and will be happy to share my experience.

As you have already discovered polybrene can be toxic for some cells. Switching to protamine sulfate may help but you might also consider using a different pseudotype such as RD114 or measles virus.

Thanks for the suggestion Michael. I would definitely consider trying that next.

Hi Naina,

In my exprience, the most efficient and less towic protocol with your type of cell is retronectine+spinoculation.

Ploybrene is often toxic...

You can have a look here for some types of protocol transduction or request more advices

https://www.geg-tech.com/knowledge/lentiviral-vectors-multifaceted-tools/transduction-with-lenti-one/

Hope this will helps you

Regards

Nicolas

For me protamine sulfate with rat endothelial cells and human corneal cells work very nice, but I want to try LentiBOOST, has anybody experience with it?

Regards,

Emilio

Hi Edwin,

You can also test Lenti-ONE which are ultra-concentrated and, for example, which induce up to 80% of positives CD4+T.

I can forward you some data about the efficiency of this vector in several different contexts if needed.

You can email me at [email protected]

Regards

Nicolas

In continuation to Naina's query, I would like to know ifProtamine Sulfate work equally well with Retroviral transduction as it does it Lentiviral protocols?

For a long time, I had been trying to transduce Heamatopoietic progenitor cells with retroviral supe obtained from transfected Plat-E cells cells, in presence of Polybrene (10ug/mL supe final concentration). I've had poor transduction efficiencies most of the times.. any suggestions?