I need to transfect plasmid DNA into Jurkat cells, but it seems that these cells are hard to transfect. If anyone has an experience, can you share it with me?
Lentiviral transfection is a good, efficient way to go. The only issue I've had is that sometimes I get an immune response from the cells, which is difficult to account for in research. I tend to use electroporation (with this buffer: https://altogen.com/product/jurkat-electroporation-kit-t-cell-leukemia-tib-152/) and it's worked well so far.
Essentially this protocol: http://www.med.upenn.edu/pennimig/user_images/Transfection%20of%20Jurkat%20T%20cells%20by%20electroporation.pdf
Works for me, but using a very old basic biorad electroporator.
Obviously Amaxa nucleofector solutions and machines work very well too, but that is a more expensive way of approaching it.
William, thank you for suggestion.
For those who are following the conversation: Tried to electroporate using Amaxa nucleofector basing on this protocol: http://bio.lonza.com/fileadmin/groups/marketing/Downloads/Protocols/Generated/Optimized_Protocol_39.pdf Worked well.
Hi Valentina,
the Neon transfection system form Life Technologies is another option. Compared to Amaxa, the expression levels of transfected proteins are a bit lower but transfection is more homogeneous and viability is better.
http://www.lifetechnologies.com/fr/fr/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system.html
Anyway Jurkat can also be transfected with standard cuvette electroporators (e.g. BioRad). Depending of yor plasmid you may get 50% or more transfected cells.
Good luck,
Vincenzo
Yes, indeed, the transfection by amaxa is quite heterogenous and the death rate is quite high, but the result is satisfying as about 70-80% of cells are expressing the gene. For those who has a choise, maybe Neon transfection would be more accurate (I just use what I have in my lab).
I'd highly recommend trying Xfect, chemical transfection reagent, as it is far better than any of the other chemical transfection reagents we tried. Neon and Lonza electroporation also work.
If you have access to a Neon electroporator, give it a shot: https://www.thermofisher.com/uk/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-transfection-system-information.html
https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/cell-culture/neon-protocols.par.97686.file.dat/jurkat-microporation.pdf
Hi!
3 years have passed since the start of the conversation. Seems that I can add some comments. The best result I've achieved using lentivirus. Good efficiency and viability, plus you create a stable line so you don't need to transfect cells again and again. For Jurkat there is a special method for lentiviral transduction called spinoculation. I simplified this method by just spinning the cells with the virus in 15ml tubes 30 min 800g 32C in swing bucket rotor. Afterr that suspended and plated without changing the medium.
Before I started to transduce, I also tried the TransIT-Jurkat transfection reagent from Mirus. It was very efficient, but all the transfected cells died. Maybe some condition was wrong (as medium, serum etc), I didn't start to waste the time on it. Lentivirus worked well)
Hi Valentina,
Thanks for sharing your spinoculation protocol.
What is the total volume in which you spin the cells?
Do you include a transfection reagent or the vikus just gets in more easily due to the centrifugal force? Thanks
Lentiviral transfection is a good, efficient way to go. The only issue I've had is that sometimes I get an immune response from the cells, which is difficult to account for in research. I tend to use electroporation (with this buffer: https://altogen.com/product/jurkat-electroporation-kit-t-cell-leukemia-tib-152/) and it's worked well so far.
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