Dear Shreya,

Rat and mouse pyramidal neurons are pretty similar when it comes to transfection. Could you specify the method you use, the age of the neurons at the time of transfection and roughly the protocol you use.

Cheers,

Dear Sebastian,

I use the optifect-lipid-based method to transfect the primary neurons at DIV 7 and DIV 14. I transfect them using a serum-free medium "Neurobasal" for 6 hours and change the medium. I fix them at DIV 8 and Div 15 using 4% PFA and proceed with immunostainings. I use cortical and hippocampal neurons.

Thanks

Dear Shreya,

I'm not familiar with the optifect-lipid-based method. I typically use lipofectamine to transfect attached neurons although its efficiency in the conditions you mention would be only around 5-10% for both mouse and rat hippocampal neurons.

Somehow, however, rat neurons seem to be more resistant to lipofection than mouse neurons. Could it be that in your transfection conditions mouse neurons are just dying out? Can you borrow lipofectamine to test transfection in rat and mouse neurons using the same DNA concentration and times you used with the optifect kit. This would be to rule out whether the optifect kit may somehow affect mouse neurons more than rat ones.

Also you can consider lowering the time of exposure to the liposomes (4h instead of 6, or you can keep the 6 h but reduce the liposome concentration) and keeping the DNA concentration low (not higher than 5 ug total).

A further test I would do is, in your present conditions, fix the cells 4-6 h after media change and check for cell undergoing apoptosis vs a mock transfected (no liposome-DNA mix) control.

I'll let you know if I think of something else.

Good luck with your transfections.