Brain Cell Isolation from Zebrafish Larvae: A Technique to Obtain Intact Neurons, Macrophages, and Microglia from Zebrafish Brain Tissue

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.

Add 1.5 milliliters of 15 millimolar Tricaine per 50 milliliters of medium to 50 larvae to prepare anesthesia. Then, use a 3-milliliter Pasteur pipette to transfer 10 larvae at a time into a 55-millimeter Petri dish filled with ice-cold E3 medium with Tricaine to terminally anesthetize them.

Under a stereomicroscope, align 10 larvae in the center of the Petri dish. Then, using surgical scissors, transect the larval heads above the yolk sac.

With a 3-milliliter Pasteur pipette, take up all the heads, and with as little liquid as possible, transfer them into a glass homogenizer on ice containing 1 milliliter of ice-cold Media A.

Replace each small Petri dish containing ice-cold E3 plus Tricaine with a new one every 30 minutes to assure that transection is performed in cold E3 plus Tricaine medium.

Replace the ice-cold Media A in the glass homogenizer when the color starts fading. Once the entire group of heads has been collected, remove all the Media A from the glass homogenizer and replace it with 1 milliliter of fresh ice-cold Media A.

With the homogenizer still on ice, use a tight-fitting pestle to disrupt the brain tissue by performing 40 rounds of crushing and turns for 3 to 5 dpf larvae and 50 rounds for 7 and 8 dpf larvae.

Then, add 2 milliliters of Media A to the cell suspension to dilute the cells and reduce their agglomeration with myelin. Eliminate cell agglomeration by running the cell suspension through a 40-micron cell strainer into a cold 50-milliliter falcon tube on ice. Repeat this step three times.

Transfer 1 milliliter aliquots of cell suspension into cold 1.5-millimeter tubes, and spin them at 300 x g and 4 degrees Celsius for 10 minutes. Then, using a 10-milliliter syringe with the 23G x 1" needle, remove the supernatant.

With 1 milliliter of ice-cold 22% density gradient medium, gently overlaid by 0.5 milliliters of ice-cold 1x DPBS, gently resuspend the cell pellet. Spin the tubes at 950 x g without a break and slow acceleration at 4 degrees Celsius for 30 minutes.

After the spin, discard the DPBS density gradient medium and myelin trapped at the interface. Then, use 0.5 millimeters of Media A with 2% NGS to wash the cells and spin the tubes at 300 x g at 4 degrees Celsius for 10 minutes.

Remove as much supernatant as possible, then pull the cell pellets from the same experimental condition together in 1 milliliter of Media A with 2% NGS.

If the cells of interest express a fluorescent protein, run the cell suspension through a 35-micron cell strainer cap and transfer them into cold 5-milliliter FACS tubes on ice protected from light.