Hello, everyone!
Today I got Amaxa Nucleofector II for my work. I haven`t read all the manual yet, but as far as I could find out, there are different kits for almost all cell types. I got a legacy kit for hMSC, but my work includes nucleofection of primary T cells, human, sometimes murine. Therefore I need to ask, can I use one kit for other cell type or I need to buy a new one?
Also, are there any buffers I can make myself? It is very expensive and timeconsuming to buy one where I`m working. Does anyone have any experience in that area?
Hello Maksim,
Yes you can make your own buffers for the Amaxa nucleofector electroporator, and this method is much more cost effective. Additionally, there are different kits out there, as well as Amaxa nucleofection protocols that are programmed on the instrument, that are optimized for different cell types (even between the same cell type that are treated differently, i.e. activated and unactivated T cells).
In addition to the in-lab made buffers, you could also use generic nucleofection chambers.
I've linked two manuscripts below, one by Chicaybam and the other is from our lab testing some of these buffers. Although I chose lentiviral based transduction of T cells as my preferred method, electroporation can also be useful. I recommend that you make the buffers and test all of them on your purified T cells. I haven't worked with the other cell types you mentioned, so you may need to try alternative Amaxa programs for those specific cell types. You can get a general idea about the specific program from Lonza.
http://www.ncbi.nlm.nih.gov/pubmed/26027856
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060298
Hope this helps,
Mahmood
Martin,
Thank you for your input. The transfection buffers listed in the manuscript are very useful. I've linked a few people to your protocol and it worked very nicely for B cells, and Jurkats, HuT78, and primary T cells. Also, With a slight modification in one of your buffers (addition of glucose, l-glutamine, and reduced Glutathione)we were able to produce >95% transfection efficiency (seen via flow cytometry and the bright green cell pellet) on HBE cell lines with about 30% cell death. However, I haven't tested this buffer in other cell lines.
Regards,
Mahmood
Thank you, Mahmood and Martin. I`ll definitely examine all those articles. We tried to use that kit we got. At virtually no problem we got 40% transfection efficiency with 50 to 70 per cent survival with primary unstimulated human CD4 T cells. I think now we will try links you provided.
Great thread thank you for the above information Martin, Maskim, and Mahmood
Please care to share the buffer recipe and protocol if any of you have tried for mouse primary T cell unactivated via electroporation and program setting for Amax nucleofector electroporator. Thank you.
Mahmood can you share the component you have modified in Martin protocol to get 95% transfection efficiency and at what concentration.
THank you all
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