Without polybrene, the transductioin efficiency is very low in my primary tumor sphere primary cells
Two thoughts: 1) Concentrate your virus to increase your titer, and 2) try a spinfection (spinnoculation). For the spinfection, add virus to your cells in a relatively small volume. Spin the plate in a swing bucket rotor at 1000 g for 1 hr at room temperature. Both of these should increase your infection efficiency.
Also, what concentration of polybrene are you using? Standard is 4 - 8 ug/ml.
I agree with the previous responses- try to increase the viral titer and try spinoculation. I've worked with some cells for which polybrene is toxic. I've also found that doing multiple rounds of infection can improve my efficiencies. I'll usually do one round of infection initially and if I have low efficiency, then I'll do one to two additional rounds. If it's still low, I'll go through selection using antibiotics or FACS. Good luck!
I second the use of spinofection. In addition, I have had some success with using Pluronic F108 in addition to or instead of polybrene (as described in this paper: http://onlinelibrary.wiley.com/doi/10.1002/jgm.2653/abstract Generally, at 1mg/mL of F108 I have not had any issues of toxicity. Note: If you try F108 together with polybrene, do not premix the solutions, then they stop working. Just add each stock to the virus/cells directly.
Hi Hong,
Try lentiviral vectors with ultra-high titers without polybrene or others adjuvants which have negative effects.
My lab is able to produce this type of vectors, indeed we have had several project needing the transduction of very refratory cells such as primary cells and thanks to our strong know-how in LV vectorology (+15 years) we are able to produce ultra efficient LV which allowed to go ahead in these research projects which for some were blocked for several months.
Feel free to contact me if you want dicuss about that
Regards
Nicolas
www.geg-tech.com
Dear Hong,
I think an useful alternative could be DEAE. Although I haven't infected PBMCs but have worked regularly with T-cell lines such as Jurkat, SupT1 etc where I have had pretty successful lentiviral infections with an optimal of 25 ug/ml DEAE as final concentration during infection. After 6h of infection, you need to wash the cells and replenish with complete media (mostly RPMI). You may leave the infected cells in DEAE-media to a maximum of 12 h but not beyond.
Hope this works..
Best,
Sutanuka
I used 8ug/ml polybrene for infecting iPSC with lenti-viruses. The Pb alone in medium can be very toxic to some of my iPSC lines, however with appropriate amount of viruses the toxic effect is hugely reduced. You could titrate the amount of virus on cells seeded at various densities (e.g. 40%, 60% and 80% confluent), with and without the presence of Pb, to find out what is the best combination for you.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line