Dear Pablo,

I can recommend you another transfection method for mature neurons that has been proven and largely published.

This is called Magnetofection and the reagent is NeuroMag, it can efficiently transfect primary neurons from 1 to 22 DIV. You can see information and publications on the website:

http://www.ozbiosciences.com/transfection-cell-specific/52-neuromag-neuron-transfection-reagent.html

Here is one example: "High transfection efficiency on primary dopaminergic neurons at 21 DIV."Underhill SM et al, Neuron. 2014

Article Amphetamine Modulates Excitatory Neurotransmission through E...

If you need more information and protocol please do not hesitate to contact me at [email protected]

Good Luck

Hi Pablo!

I was wondering if you find a solution to this problem. I'm facing the same issue and I was wondering if there is protocol out there using lipofectamine in mature neurons. I get amazing results from DIV4-10.

Hi Juan,

What I figure is that the neuron must be cultured in Neurobasal plus and B27 plus, no antibiotic or any other reagent. They resist very well up to 21 DIV.

Then when I have to transfect I remove the medium and save them for later and put 0.25mM of acid kinurenic or other NMDA blocker on it. The medium of transfection I am using MEM (with 0.12 mM of acid kynurenic) and I transfect for one hour, then I put back again the conditioning medium. This seems to work very well, although still with low efficiency with lipofectamine , I got between 1-5 neuron transfected when I plate 100 000, that is the only problem.

Hope it helps

Cheers

Thank you so much Pablo! I'll try this and other tweaks and will update the thread!

Cheers!

Juan

Good luck. For coverslips of 24 well I am using 1 ug DNA and 1.5 uL of lipofectamine 2000 for the reaction I use also MEM