I need to transfect siRNA into Jurkat cells, If anyone has an experience about which transfection reagent is best? I have already tried for EGFP using transfectamine 3000, but could not get any transfection...I would be very grateful if anybody could help.
… Read more Answer this question
I need to transfect siRNA into Jurkat cells, If anyone has an experience about which transfection reagent is best? I have already tried for EGFP using transfectamine 3000, but could not get any transfection...I would be very grateful if anybody could help.… Read more Answer this question
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.
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.
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
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
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).
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).
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! 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)
I need to transfect siRNA into Jurkat cells, If anyone has an experience about which transfection reagent is best? I have already tried for EGFP using transfectamine 3000, but could not get any transfection...I would be very grateful if anybody could help. … Read more
Answer this question
I need to transfect siRNA into Jurkat cells, If anyone has an experience about which transfection reagent is best? I have already tried for EGFP using transfectamine 3000, but could not get any transfection...I would be very grateful if anybody could help.… Read more
Answer this question
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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.
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.
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VOTE
Adding back william grey's protocol to keep this discussion complete. It appears to have been lost
Adding back william grey's protocol to keep this discussion complete. It appears to have been lost
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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
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
More
VOTE
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
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
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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
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
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VOTE
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).
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).
More
VOTE
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!
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)
More
VOTE