Does your virus have a selectable marker such as puromycin or hygromycin? They usually do, so you add your selection within 24h of transduction and the untransduced cells will not grow and die.

Gregory Dressler Yes, i added antibiotics for selection but all of them died. So, i am not sure what went wrong with the transduction.

Dear Narmadha Murugesan

The optimal cell density for transducing C2C12 cells with lentivirus is typically when the cells are at a subconfluent state, ideally less than 50% confluence. This allows for efficient viral entry and reduces cell-to-cell contact, which can hinder viral infection. For a 6-well plate, seeding around 30,000 to 50,000 cells per well 24 hours before transduction can help maintain an optimal density at the time of transduction (Tapia O., Methods Mol Biol. 2016).

Now regarding Lentiviral transduction and selection process with antibiotics, I would recommend two options:

■ 1. Choose the correct antibiotic (ab) concentration: prepare a cell plate (24-well plate) and add increasing doses of ab. 48H to 72H after observe what minimal dose is lethal for your cells and work with that. Depending on the cell line and culture condition it may vary a lot.

■ 2. Try our LentiBlast Premium Transduction enhancer to increase viral infection.

LentiBlast Premium is a patented modified poloxamer that we developed a couple of years ago and that acts by simultaneously neutralizing electrostatic repulsions between membrane and viral particles and enhancing viral fusion with cell membrane. Due to a favorable “membrane permeable effect” limiting the transmembrane potential changes, it is non-toxic and totally compatible with cell viability.

It is a real asset to viral infection because it allows to increase the effectiveness of the viral vector, thus reducing the MOI, and consequently the immune response of the cell. Secondarily, it reduces the cost of experience. And thus I believe it would be ideal in your experiment.

LentiBlast Premium was demonstrated to be efficient of multiple cell types from Hematopoietic Stem Cells, to T lymphocytes and I am sure it would be worth give it a try for your experiment.

Some examples of publications with LentiBlast Premium:

• HEK293: Brown AL. Sci Rep . 2021 Oct 25;11(1):21008
• Bone marrow derived macrophages: Fonseca GJ. Nat Commun. 2019 Jan 24;10(1):414.
• Hematopoietic Stem Cells : Burton OT. Immunity. 2024 Jun 11:S1074-7613(24)00277-2.
• HMEC : Nassour J. Nature . 2023 Feb;614(7949):767-773.
• Myeloid progenitor : Verdys P. EMBO J. 2024 Jul 18. doi: 10.1038/s44318-024-00173-7.
• T lymphocytes CD4+ : Claireaux M. Nature Commun 2022 Jan 26;13(1):521.
• THP-1 : Chirayath TW. Nat Commun. 2024 Sep 18;15(1):8179.

you can have more infos at: https://ozbiosciences.com/lentivirus-retrovirus/113-418-lentiblast-premium-transduction-enhancer.html#/12-capacity-500_l

Would you like to give LentiBlast a try, please do not hesitate to contact me via ResearchGateor directly at: [email protected]; I will be glad to send you a sample !

best regards,

Cedric

Hi Cedric!

Sorry, i did not notice your response.

Thank you so much for sharing your insights.

I will read about Lentiblast which seems to be interesting.