Details for amaxa FAQ article #7

«amaxa suggests using 4-5 million neural cells per 100µl nucleofection® reaction. Why is such a high cell number required? How can I perform nucleofections with fewer cells ?»

We recommend preparing and transfecting a "mixed glial culture". This provides you with enough cells without having to isolate pure neurons. By using this "high" amount of cells during nucleofection and plating them as densely as recommended, you will increase cell survival significantly. The glial cells support neuron recovery after nucleofection (except for adult mouse DRGs which are typically not supported by glial cells).
By changing the culture medium to a medium that impairs glial cell growth you can start depleting these cells 24 hours after nucleofection to get a pure neuron culture.
Using our 96-well Shuttle System, 250,000 cells and less will suffice per nucleofection sample. Even lower numbers are now possible using our new Basic Neuron SCN Nucleofector Kit, which facilitates the nucleofection of less than 100,000 cells per sample. However, it is still important that seeding density after nucleofection is sufficient, e.g. by seeding the cells onto 96-well culture plates or coated small cover slips.