I am currently working with HT-22 cells by transfecting them with bacterial plasmids to perform IHC. However, we have not had much success with reaching a 50% transfection rate. I usually do about a 40 hour incubation post transfection. I can not allow the cells to incubate any further because they will die due to the lipofectamine. Last week we tried a 60 hour incubation post transfection to have a better transfection rate. Unfortunately, we did have more transfected cells but many dead cells as well which defeats the purpose of our transfection. The one thing that did help us get a higher transfection rate was adding more plasmid, lipofectamine and P3000 to our cells. Does anyone have suggestions on how to improve the transfection rate without exceeding the 40 hour incubation?
P.S. I have attached files related to the products I use for my transfection and the protocol.
You could try a RCR mini-plasmid system to remove the backbone before plasmid extraction. This would generate smaller plasmids and therefore increase the transfection efficiency slightly.
I would try other transfection vectors (TransIT, jetPei, ....) there is a lot of them, change the DNA/lipofectant ratio... but there is cells that never transfect well.... tried transduction with lentivirus ?....
Kc Chico Hello. I am not sure. I will ask my postdoc. Do you happen to have any recommendations for better products?
Melissa M Torres If your protocol is fairly rigid and doesn't allow you to switch, then adjusting your Lipofectamine concentration as low as possible can sometimes help. For alternatives, both Fugene 6 and Transfectamine 5k have comparatively better post viability readings for a lot of cell types. Neuronal cells are no joke, so crossing fingers for good results for you and your team!
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