Isolation and Culture of Primary Neurons and Glia from a Rat Urinary Bladder
Transcription
Take a euthanized rat.
Open the abdomen, excise the bladder, and immerse it in buffer.
Cut open the bladder and remove adherent fat and tissue debris.
Transfer the bladder to a tube containing buffer, centrifuge, and remove the debris.
Place the tissue in a solution containing collagenase and mince it. Add more collagenase to degrade the extracellular matrix, loosening the cells.
Centrifuge and discard the supernatant. Add trypsin to disrupt the cell-cell junctions, releasing the cells.
Add rinse media containing proteins to inactivate trypsin, then centrifuge, and remove the supernatant.
Resuspend the cells in media; filter them to remove aggregates, and obtain a single-cell suspension.
Centrifuge and remove the supernatant. Resuspend the cells in media.
Plate the cells on coated coverslips to facilitate neuronal and glial cell attachment. Remove the non-attached cells.
Add neuron media containing growth factors to promote cellular maturation, establishing a primary neuronal and glial culture.
