Hello Sunyna Saun

Protocol in brief:

1. After having added lentiviral vector to HaCaT cells, incubate overnight at 37°C / 5% CO2.

2. Change medium the next day. (Please note: If desired, lentiviruses can be removed 4hrs after inoculation, as most lentiviruses will have adsorbed to the cells within a few hours).

3. Assay cells at 48-72hrs post transduction. Add puromycin-containing growth medium to cells at 48hrs post transduction. Optimal puromycin concentration for HaCaT cells is 2.5μg/mL. If you aren’t satisfied with the mentioned puromycin concentration, you may perform the “kill curve” for puromycin and obtain the optimum concentration for puromycin.

4. Replenish puromycin-containing medium every other day. Puromycin will kill non-transduced cells in 2-3 days.

5. To generate stable cell line, continue selection process for 1-2 weeks post transduction.

It is good that you have added polybrene as it is a cationic polymer that acts by neutralizing the charge repulsion between the virus and the cell surface (both negatively charged), thus increasing the transduction efficiency. But instead of using 1µg/ml polybrene, you may increase the polybrene concentration to 5µg/ml (the range of polybrene used is 3-10µg/ml).

Good Luck!

Thank you Malcolm Nobre

Dear Sunyna Saun

A possible cause to this may be the fact that transduction rate of your keratinocyte cell line HaCat is really low, meaning that as soon as the antibiotics acts on them as if no virus was present. I would recommend two points:

■ 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