Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from a Mouse Embryo

Take a transgenic pregnant mouse and surgically remove the uterus with embryos containing fluorescently labeled motor neurons.

Transfer the uterus to a dish with an ice-cold buffer under a fluorescence microscope.

Use visible light to isolate the embryos.

Remove the face and tail of the embryos and orient them to a prone position.

Using the fluorescence signal, make an incision at the midbrain to expose the oculomotor and trochlear nuclei, which contain motor neurons.

Isolate the midbrain containing the nuclei.

Open the dorsal side of the hindbrain and spinal cord. Isolate the spinal cord, excise both ends, and collect the columns containing spinal motor neurons.

Treat the collected tissues with enzymes to dissociate the tissue matrix. Harvest the single cells and isolate fluorescently labeled neurons using FACS.

Add a culture medium to the isolated neurons, transfer the cells onto a culture substrate-coated microplate, and incubate, allowing the cells to adhere and grow.

Maintain the motor neurons in culture.