We have recently started having trouble with our mouse primary hippocampal neuronal cultures - they are much less robust and almost 100% of the neurons die in response to Lipofectamine 2000 transfection. We have used Lipofectamine 2000 extensively in the past to great success, but something has changed. Both pure neuronal cultures and astrocyte-neuron Banker cultures have the same problems. (We dissect at P0, plate in Neurobasal A with B27, Glutamax and Pen/Strep - transfections are at 6-10 DIV, transfection medium is on neurons for 3 hours, both 0.4% and 0.2% Lipofectamine 2000 cause neuronal death).
Does anyone have any insight on what may be going on?
We have heard rumors that either the B27 or Neurobasal A is bad these days. Is anyone else having the same problems?
Can anyone suggest good alternative reagents for primary neuron transfection? Any recommendations on a supplier of Calcium Phosphate transfection reagents?
Thanks!
I have tried lipo2000 diluted in neuronal culture medium (neurobasal+B27+ Glutamax + Pen/Strep) for 1h or 2h, then change with fresh medium. About 10% neurons could be transfected, and keep 60% survival rate in the next 3 days. Electroporation method could bring higher successful rate but lower survival rate.
If you have access to a Neon electroporator (invitrogen), it might be the easiest way. We have abandoned Lipo2000 completely since we started using this. Only drawbacks are the expensive consumables and the transfection at 0DIV. Hope this helps
Hi, Liang Hu! I wonder which day did you transfect the plasmid DNA into primary neurons? Thank you!