I transfected mature neuron cells by lipofectamine 2000, DIV 13 and staining at DIV 16. But, unfortunately, there was some positive transfected cells show abnormality, such as green fluorosence only like spot around cells. I wondering it was death cells. Is lipofectamine not suitable for neuron?
I read some paper use this method to observe dendritic spines and they are succeeded do this method.
Anyone can help me?
Regards,
Ria
Neuronal cells can be picky about the transfection reagent and conditions during transfection itself. Use a reagent designed for primary cells and neurons (https://altogen.com/product/altofect-transfection-reagent/) and that will prevent cytotoxicity. Usually, effectiveness is inversely correlated to cell viability; higher transfection efficiency comes at the expense of cells. However you can still get good results by using more gentle approaches tailored to primary cell cultures.
Dear Ria,
normally there are no problems because of lipofectamin transfection. Maybe your target you put inside your cells results in cell death. So did you tried to infect the cells with lipofectamin alone? If you to that and the cells also died, the problem is the lipofectamin, if they don't die the problem is the target.
Dear Sven,
Thanks for your suggestions. Now I tried that strategy to use only lipofectamine. I am still waiting 48h after treat the cells to see the results.
Hi Ria, I have read about doing this as well but never gathered the courage to do so. As you know growing healthy neuronal cultures for long periods of time is challenging so I never wanted risk it. I prefer to nucleofect b efore plating. This results in a lot of death but at least the ones that survive are efficiently transfected.
However, to answer your question try using less DNA perhaps ? That has helped me in the past with unhealthy lipofections.
Best of luck.
Hi Raj, thanks before. But, regarding you transfect the cell before plating, I wondering about the efficiency, did cell still growth well?
Actually, I`ve experience to did electroporation, then culture neuron in long time, but the cells are really2 look unhappy. Thats why, I changed my method to lipofectamine method.
But, again thanks a lot for your advice. trying less DNA, I`m not yet try it. Thanks.
Hi RIa, Lipofectamine is working well in primary neurons except you should optimize the ratio of Lipofectamine and DNA in your hands. In primary neurons, it usually takes less amount of them than the protocol suggested. And also you should always have a control group which transfect with some sequence you know will not affect your cells.
Good luck!
thanks Jing.
Now, I've tried reducing amount of DNA and lipofectamine. Hopefully, the cells will give an excellent result.
Thank you.
Hi Ria, I was wondering whether you managed to improve you transfections with these suggestions. I am trying to transfect 3-week old mouse neurons with lipofectamine 2000 but my cells keep dying after transfection.
Thanks!
Hi Marc,
Have u tried transfect neuron at young culture, such as DIV4 or DIV3 and keep the cell in ur desired culture time? I succeeded transfect my culture neuron using this method.
Maybe you can try it.
Good luck!!
Hi Ria,
I have never tried transfecting them at a younger age, mainly because I dont want to express my protein for more then a few days and my follow up analysis needs to be on mature neurons. I think I will try using an AMPA R inhibitor to limit excitotoxicity and see if that helps.
Thank you for your reply!
Neuronal cells can be picky about the transfection reagent and conditions during transfection itself. Use a reagent designed for primary cells and neurons (https://altogen.com/product/altofect-transfection-reagent/) and that will prevent cytotoxicity. Usually, effectiveness is inversely correlated to cell viability; higher transfection efficiency comes at the expense of cells. However you can still get good results by using more gentle approaches tailored to primary cell cultures.
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