I transfected H9C2 cells with different transfection reagents such as Lipofictamine 2000 and 3000, Viromer yellow and red. The best option for me was Viromer red. At the beginning the transfection it worked well but last ones failed. This is true that I got a new branch of the transfection agent, so I could be the reason. I checked it and found out the reagent to work with another plasmids. I checked plasmids, may be it happened because of plasmids degradation but they were fine. Does someone has an experience with that? Thank you in advance.
I had the same experience with the primary hepatocytes. I tried lots of different reagents (even many more than you mentioned) but Viromer red is the only one that worked at first but there was no reproducibility when I used it again. I don't have any experience with H9C2 cells but what worked for me was lentiviral transductions. So if possible I would suggest to use the lentiviral vector and there is a high probability that it will work.
Hope this helps!
Best regards,
Abhijit
Dear Sofya,
It is true that there are many reasons for a transfection not to work moreover, H9C2 cells can be considered as hard-to-transfect (myoblasts).
There are several points that need to be taken into consideration, and I'm sure we wil lfind something to enhance the transfection in your cells.
Let's go back to the points you've mentionned : typically conditions seem to have changed between the good transfections and the bad ones. This may come either from a loss in efficiency of your transfection reagent, from the plasmid, from the cells, or from the conditions.
I would highly recommend several options :
a. Change your culture conditions: use an endotoxin and contaminant free medium for culture and complexes formation. Any trace of endotoxin can impair the efficiency of transfection. Be sure that plasmid is still active by transfection on control cell line (such as HEK293).
With passaging, cells may differenciate and what you observed at the first time may not be relevant the second time. Be sure to use low passaged cells to be fully reproductible.
NOTE: It is highly important that complexes are formed in medium w/o any supplement (serum, antibiotics, proteins, growth factor....).
b. the second option would be to use Magnetofection technology to enhance the efficiency of lipofectamine in these cells. For example, Combimag (magnetic nanoparticles) reagent can be added to Lipofectamine to enhance its efficiency without changing the conditions.
c. the third option would be to try an alternative to Lipofectamine such as DreamFect or DreamFect Gold: these transfection reagents are biodegradable and have high compaction level of nucleic acid and allow using less DNA: viability is enhanced:
If you want some supplemental info or try samples of the reagents mentionned above, you can contact me at: [email protected], I will be more thant happy to help.
Good luck with your experiment,
best regards,
Cedric
MAGNETOFECTION
*** The Magnetofection technology uses a magnetic field to attract and concentrate complexes of magnetic nanoparticles and nucleic acid onto the cell surface as demonstrated by Grześkowiak BF et al, Pharm Res. 2014 Jul 18. (http://www.ncbi.nlm.nih.gov/pubmed/25033763).
http://www.ncbi.nlm.nih.gov/pubmed/25033763
Dear Cedric, thank you very much for your answer. You have mentioned that the efficacy of the trancfaction can be effected by passege. Do you have an idea why it could happen?
Dear Sofya,
you are welcome,
I must admit that I do not know for sure the process involved in such phenomena. It is based on what I have read and on observations. You can refer to the tech bulletin produced by ATCC (in attached files) ; maybe you will find usefull information.
Based on my experience, depending on the cell type, the transfection efficiency is really affected with high number passages (easier, or non permissive at all, or susceptibility for apoptosis... every schemes are observed). For primary cells, passage number alter their differentiation process ; and when dealing with HUVEC that are programmed to die, it is not recommended to use them after P2 or P3...
so definitively passage have an effect on cell behaviour, and thus on transfection.
I am sure, there will be specialists of this that would give a more detailled answer.
Best regards,
Cedric
Transduction with a lentiviral vector is probably the best solution. We've done it this way, and it works better than transfection.
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