Isolation and Culture of Primary Embryonic Mouse Midbrain Dopamine Neurons

Take midbrain floor tissue isolated from the ventral region of the midbrain of mouse embryos.

The tissue is rich in developing dopamine neurons, which release the neurotransmitter dopamine.

Add an enzyme solution to break down the tissue matrix, thereby loosening the cells.

Add a serum solution containing DNase I and mechanically dissociate the digested tissue to generate a cell suspension. The serum proteins inhibit the enzymes, while DNase I degrades extracellular DNA released during cell lysis, preventing cell clumping.

Let the tissue debris settle and transfer the suspended cells to a fresh tube.

Centrifuge and remove the supernatant, then resuspend the cells in a dopamine neuron medium, or DPM.

Take a microplate coated with a cell culture substrate at the center of the wells, creating micro-islands.

Transfer the cells to the middle of the micro-islands to culture them at a high density.

Incubate, allowing the cells to adhere to the micro-islands.

Fill the wells with DPM and incubate, facilitating the growth of dopamine neurons.

To set up primary embryonic midbrain cultures from mouse embryonic day 13.5 mouse embryos, pool all of the harvested mid-brain floors into the same 1.5