Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells
Transcription
Take a mouse embryonic carcinoma cell suspension capable of differentiating into various cell types.
Seed cells on a hydrophobic culture dish containing media and retinoic acid.
Incubate. Hydrophobic surfaces prevent cell adhesion, causing cells to form aggregates.
Retinoic acid enters cell aggregates, triggering signaling pathways and causing differentiation into neuronal progenitor cells.
Post-incubation, collect the cell aggregates.
Discard the media.
Add media containing retinoic acid and seed the aggregates onto a hydrophobic culture dish.
Incubate. Retinoic acid facilitates sustained cellular differentiation into neurons.
Collect the aggregates and remove the media.
Add a proteolytic enzyme to disrupt cell-cell adhesions within the aggregates.
Add media containing serum to stop the enzymatic activity.
Mechanically dissociate the aggregates, forming a neuronal suspension.
Centrifuge and discard the enzyme-rich supernatant.
Resuspend the neurons in media and seed them on an adherent multi-well plate containing media.
Incubate. Neurons adhere to the plate and mature, extending long projections.
