I would like to know if any of you have ever worked with H9C2 cells and successfully transfected them. Alternatively, has anyone worked with any type of cardiomyocytes and performed a successful transfection?
We're trying to transfect them with pCMV plasmid containing our desired gene, which we later observe with confocal microscopy. The efficiency is extremely poor (so far only 3 cells had observable expression!). If you have experienced problems with transfecting and have any solutions to increase the yield, we would be very grateful. Everything is done according to the protocol, but we didn't experiment with DNA:Lipofectamine ratio, however I doubt it's the root of the problem.
We're currently trying to establish what's wrong using CHO cells, but I would be more than happy to get the cardiomyocytes working. H9C2 are 17th passage, maybe this can influence their lipofection efficiency?
Our lab had trouble transfecting H9C2 cells with Lipofectamine 2000 as well.
Our collaborators suggested FuGENE HD (Promega) to transfect H9C2 cells, and we've had much better success and transfection efficiency with that.
I was doing some very successful transfections with Lipofectamine 2000 in H9c2 cells but suddenly it has stopped working. I am trying all sort of things including checking plasmid integrity. I might give FuGENE HD a try too.
Just a quick update, transfections are working again with Lipofectamine2000.
We also have a problem using Lipofectamine 2000 to transfect H9c2 as well. We did successfully transfect H9c2 with pCMV-GFP once, last few weeks, but it hasn't worked sine then. We try to repeat the experiment for 4 times, but failed. We've found that cells were died after 24 hours of transfection.
I have used Lipofectamine 2000 with H9C2s with a ratio of 6:2 for 24 hours and it worked fine. Make sure you remove any antibiotics from the media 24 hours before transfection and the cells need to be at a confluency of around 70%. I do recommend to check different ratios and time of transfection as well as the confluence of the cells. Good luck.
Another thing. It is important to note that H9C2 are myoblasts from rat heart. They can be differentiated to skeletal muscle or cardiomyocytes by changing the media to 1%FCS (skeletal) or 1%FCS + Retinoic Acid (cardiomyocytes) for 4-6 days. There are differences between them so make sure you have the right stage of the cells. They can be transfected with Lipofectamine 2000 at any stage.
Evelyn, is your 6:2 ratio for Lipofectamine:DNA respectively, or the converse (DNA:Lipofectamine)?
I have undertaken five luciferase assays now with my H9C2 cells, passage 16, using a 3:1 lipofectamine:DNA ratio, and only one of them was moderately succesful. The other four had odd values popping up here and there...
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