Hello there! I have some query to solve regarding my lentivirus infection! I have successfuly generated lentivirus particles by 293T transfection. And i have used this supernatent to infect 293T cells, but i am unable to get clear GFP signal (marker). so my questions are; 1: what is the best way to cencentrate lentivirus? can i use centricon (if yes what's the optimal pore size? 2:how to increase infection efficiency? is there some reagent i can use which is compatible to 293T cells?
You can do ultra centrifugation to pellet the virus and resuspend in the desired volume. If not you can use centricon with 100 kDa cut off. For other methods, follow this article.
https://www.nature.com/articles/srep13875
You can centrifuge at 20000rpm for 2hours and then resuspend in the desired volume.
In the first place, did you use VSV-G Envelope to produce your lentiviral particles? Without an appropriate envelope, you cannot infect all cells. In my experience, viruses produced from 293T cells are quite abundant and need not be concentrated unless there is a specific issue in your context.
@sanath, I used PsPax2 and PMD2G packinging plasmids along with lentivirus expression vector!
In that case, I agree with the prior suggestions to concentrate the virus, although it has not been necessary for my experiments in the past. Additionally, you can use Polybrene or Hexadimethrene bromide to enhance the infection efficiency. Spinoculation of the virus with your cells would also be a good idea to enhance infectivity, where you add viruses to your cells and spin down at 1200g for 1 h, so that the cells could uptake virus efficiently.
@Sanath, In the past i infected cells with Viral supernatent collected after 24 and 48 hrs of lentivirus transfection and infected 293T cells. Bad news is i am unable to replicate the results. that time i did not centrifuge, rather used 0.22 syringe filter and then used this filtered supernatent to infect cells.
You can use from Clontech. use the *Lenti-X Concentrator.to concentrate Lentivirus Concentration—Concentrate Lentiviral Supernatants Without Ultracentrifugation
Lenti-X Concentrator provides a fast, simple, and highly efficient method for concentrating any lentiviral stock. In the simple protocol, you just mix your lentiviral supernatant with the lentivirus concentration reagent, incubate for a short period, and spin the mixture in a standard centrifuge. You will get an immediate 100-Fold Increase in Titer.
Hi,
If you want to enhance transduction efficiency try to use (independently or together):
1. supplementation with polybrene
2. plate coated with fibronectin
3. spinoculation method
4. enhance viral particle level
5. enhance cell viability after transduction by Revitacell or ROCK inhibitor supplementation
Good luck!
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