Culturing Fluorescent Excitatory Neurons from a Transgenic Mouse Pup Brain

Take a transgenic mouse pup brain.

Remove the cerebellum and dissect the brain. Separate the cortico-hippocampal tissue containing fluorescent protein-expressing excitatory neurons.

Transfer the tissue to a culture plate containing buffer.

Mince the tissue and transfer it to a tube containing papain.

Papain digests the tissue's extracellular matrix, loosening cells, including neurons.

Transfer the digested tissue to a tube with BSA proteins, stopping the enzymatic activity and preventing cellular damage.

Mechanically dissociate the tissues, releasing cells.

Centrifuge and remove the supernatant containing debris and enzymes.

Resuspend the cells in low-fluorescence neuronal media and filter through a cell strainer, removing cell aggregates.

Perform fluorescence-activated cell sorting to identify and sort the fluorescent neurons.

Transfer the sorted neurons to a microcentrifuge tube and centrifuge. Resuspend neurons in neuronal media.

Seed neurons on poly-L-lysine-coated coverslips in a multi-well plate. Neurons adhere to the coated surfaces.

Add neuronal media. Incubate to facilitate neuronal growth.