I'm trying to transfect freshly isolated mouse primary hepatocytes with specific Amaxa nucleofection kit. Hepatocytes are viable after isolation but they die shortly after transfection and I can't detect no expression of the protein of interest neither by Western Blotting or by fluorescence microscopy (I use a GFP-tagged protein). Another major problem is that hepatocytes are autofluorescent cells and they can emitt fluorescence both in the green and red channel. How can I overcome this issue? Can I use indirect fluorescence using an anti-GFP antibody (+ secondary fluorescent antibody) to increase the specific signal versus background signal?
I also had bad experience for using neucleofection kit. I solved this problem by lipofectamine-2000. Although the transfection efficiency is not high (about 5~10%), it is enough for immunocytochemistry and hepatocytes are healthy after transfection by lipo-2000. Like you said the autofluorescence is high in hepatocytes. I resolved this problem by increase the level of expression protein. If it is difficult to upregulate protein level, like you said, I used anti-tag (GFP, RFP, MYC......ect) antibody to enhance signal. I can always get good images by either of these two way.
I also had bad experience for using neucleofection kit. I solved this problem by lipofectamine-2000. Although the transfection efficiency is not high (about 5~10%), it is enough for immunocytochemistry and hepatocytes are healthy after transfection by lipo-2000. Like you said the autofluorescence is high in hepatocytes. I resolved this problem by increase the level of expression protein. If it is difficult to upregulate protein level, like you said, I used anti-tag (GFP, RFP, MYC......ect) antibody to enhance signal. I can always get good images by either of these two way.
We found the most efficient transfection method for primary hepatocytes to be mRNA transfection. The nuclear entry step that is required with DNA is the major hurdle in these cells. With mRNA transfection, the mRNA is delivered to the cytoplasm and readily available for translation. Lipofectamine MessengerMAX is a new reagent developed specifically for mRNA based transfection and you can request a free sample on the Life Technologies website (https://www.lifetechnologies.com/us/en/home/global/forms/lipofectamine-messengermax-sample-request.html).
Let me know if you have any more questions for the mRNA transfection.
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