I'm trying to transfect the 32D cells (in suspension) by using lipofectamine 2000, but it didn't work, I will try again by Lipofectamine 3000.
Most of articles use electroporation or infection by lentivirus, I had never used that before, so if you are familiar with this kind of cells, could you help me ?
Thank you
I am not familiar with that cell type, but if it is bone marrow-derived cells, do not attach and in suspension, you might want to try lentiviral method. Previously I have done lentiviral transduction in suspension cells and it works really well. You can follow this article for the method.
http://www.ncbi.nlm.nih.gov/pubmed/24223881
I am not familiar with that cell type, but if it is bone marrow-derived cells, do not attach and in suspension, you might want to try lentiviral method. Previously I have done lentiviral transduction in suspension cells and it works really well. You can follow this article for the method.
http://www.ncbi.nlm.nih.gov/pubmed/24223881
Dear colleagues,
in light of our experience, lentiviral transfection works satisfactorily either in suspension or adherent cultures. I agree with Sugata it is an optimal and quite easy procedure for transfecting cells. Good luck!
Bone marrow derived cells are notorious for not being the most efficiently transfectable cells. The best way is to go the viral route. You can use retrovirus instead of lentivirus. It is sometimes easier to get safety protocols approved with retroviruses than lentiviruses.
Hi again,
there are some important considerations to be commented in relation to this question. Although electroporation has several advantages such as versatility (works with any cell type), relative high efficiency, very low DNA requirements, and the ability to operate in living organisms, and is most commonly used to transfect cells transiently, (despite stable transfection is possible as well) disadvantages include, among others, potential cell damage (maybe this effect is higher in tiny cells like yours) and the nonspecific exchange of molecules into and out of the transfected cell. Try both methods if possible and inform us of the results...
Good luck!!
I did the the nucleofector has a specific protocol for these cells. It may be a good idea to try this if you have one nearby. However, be ready to expect a low viability of your cells after nucleotransfection. In my experience, while the cells did transfect using this system, i did lose over 70% of them.
Hello,
Thank you Santiago and Aurelie for your nice notes which are very helpful for my experiments.
I'm going to try with electroporation on friday and retroviral infection next week, I hope it will be more successful than lipofectamine 2000.
I will let you informed by the results.
Best regards
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