Brain Cell Isolation from Zebrafish Larvae: A Technique to Obtain Intact Neurons, Macrophages, and Microglia from Zebrafish Brain Tissue
Transcrição
The zebrafish brain consists of neurons – nerve cells that play a prominent role in processing information. Additionally, it comprises a population of supporting immune cells, including macrophages and microglia, that inhabit the interneuronal space.
To isolate all these types of brain cells from a zebrafish, first, take anesthetized zebrafish larvae in a Petri dish containing appropriate media.
Visualize the zebrafish under a stereomicroscope and transect the larval heads above the yolk sac to keep the brain intact.
Transfer the excised heads into a homogenizer filled with pre-chilled media, placed on ice. Low temperature helps prevent degradation of brain cells.
With a pestle, homogenize the zebrafish brain tissue. Homogenization disintegrates the extracellular matrix and initiates the dissociation of neurons, macrophages, and microglia from the brain tissue.
This step also separates myelin – a lipid-rich insulating layer – from neurons.
Pass the homogenate through a strainer to entrap large cell clumps.
Centrifuge the filtrate to pelletize the cells and myelin sheath fragments. Discard the debris-containing supernatant.
Resuspend the pellet in an appropriate density gradient medium and overlay it with a buffer.
Spin down the tube at a low speed. The lighter myelin fragments form a distinct band at the interface of the buffer and density gradient media, while the denser cells settle at the bottom.
Aspirate the top layers and resuspend the brain cells in fresh media for further use.
