All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Homogenization
NOTE:&#160;All steps are performed 4 &#176;C.
<ol>
	<li>Add 1.5 mL Tricaine (15 mM) to 90 mm Petri dishes containing 50 larvae in 50 mL of E3 embryo medium treated with 200 &#181;M PTU to terminally anesthetize them.
	<ol>
		<li>Suck up 10 anesthetized larvae from the Petri dishes with a 3 mL Pasteur plastic bulk pipette.</li>
		<li>Transfer anesthetized larvae 10 by 10 into a 55-mm Petri dish filled with ice-cold E3 embryo medium + Tricaine.</li>
		<li>Under a stereomicroscope, align 10 larvae in the center of the Petri dish. Then, transect larval heads above the yolk-sac using surgical micro-scissors (exclude swim bladder to avoid floating heads).</li>
		<li>Suck up heads from the Petri dishes with a 3 mL Pasteur plastic bulk pipette. Wait until all heads gather within the pipette tip and then transfer them into a glass homogenizer containing 1 mL ice-cold Media A (transfer in a minimal volume to reduce Media A dilution by E3 + Tricaine). Keep the glass homogenizer on ice. Use one homogenizer per experimental condition.</li>
		<li>Replace each small Petri dish containing ice-cold E3 + Tricaine with a new one every 30 min to assure that transection is performed in cold E3 + Tricaine medium.</li>
		<li>Replace the ice-cold Media A in the glass homogenizer when the color starts fading. 
		NOTE:&#160;Media A dilution can alter the head tissue due to temperature changes.</li>
		<li>Once all heads have been collected (600 heads/condition), remove the maximum volume of Media A from the glass homogenizer and replace it with 1 mL of fresh ice-cold Media A.</li>
		<li>Disrupt the brain tissue with a tight glass homogenizer on ice. Perform 40 rounds of crushing and turns for 3&#8211;5 dpf larvae and 50 for 7 and 8 dpf larvae.</li>
	</ol>
	</li>
	<li>Add 2 mL of Media A to cell suspension (1 mL Media A/200 heads), which will dilute cells and reduce their agglomeration with myelin to facilitate their separation during the centrifugation in density gradient medium.
	<ol>
		<li>To eliminate cell agglomeration, run the cell suspension through a 40 &#181;m cell strainer placed on top of a cold 50 mL falcon tube maintained on ice. Repeat this operation 3 times.</li>
		<li>Transfer 1 mL of cell suspension into cold 1.5 mL tubes and spin them at 300 g for 10 min at 4 &#176;C.</li>
		<li>Remove supernatant using a 10mL syringe + needle 23G x 1''.</li>
	</ol>
	</li>
	<li>Resuspend the cell pellet with 1 mL of ice-cold 22% density gradient medium gently overlaid by 0.5 mL of ice-cold DPBS 1x (do not mix them, an interphase between both solutions will be seen).
	<ol>
		<li>Spin tubes at 950 g without brake and slow acceleration for 30 min at 4 &#176;C. 
		NOTE:&#160;This step separates myelin from other cells by sequestering it at the interphase of DPBS 1x and 22% density gradient medium, whereas cells will pellet at the bottom of the tube. Myelin removal is more efficient when cell concentration is not too high.</li>
		<li>Using a 10 mL syringe + needle 23G x 1''&#160;discard the maximum of DPBS, density gradient medium, and myelin trapped at their interphase.</li>
		<li>Wash cells with 0.5 mL of Media A + 2% of normal goat serum (NGS), then spin tubes at 300 g for 10 min at 4 &#176;C.</li>
		<li>Discard the maximum of the supernatant, then pool all cell pellets from the same experimental condition together in 1 mL of Media A + 2% NGS.</li>
	</ol>
	</li>
	<li>If the cells of interest express a fluorescent protein-like macrophages/microglia from mpeg1:eGFP or neurons from NBT:DsRed transgenic fish, run the cell suspension through a 35 &#181;m cell strainer cap and transfer them into a cold 5 mL FACS tubes on ice, protected from light. 
	NOTE:&#160;Alternatively, immunostaining of microglia can be performed.</li>
</ol>

<table><tbody><tr><td>1-phenyl 2-thiourea (PTU)</td><td>Sigma</td><td>P7629</td><td></td></tr><tr><td>Hepes</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G8644-100ML</td><td></td></tr><tr><td>HBSS 1X</td><td>Gibco</td><td>14170-088</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-02</td><td></td></tr><tr><td>HBSS 10X</td><td>Gibco</td><td>14180-046</td><td></td></tr><tr><td>DPBS 1X</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Tricaine (MS222)</td><td>Sigma</td><td>A5040</td><td></td></tr><tr><td>Sterilin standard 90 mm petri dishes</td><td>ThermoFisher</td><td>101VIRR</td><td></td></tr><tr><td>Surgical micro-scissors</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>3 mL Pasteur plastic bulk pipette</td><td>SLS</td><td>PIP4206</td><td></td></tr><tr><td>Glass homogenizer</td><td>Wheaton</td><td>357538</td><td></td></tr><tr><td>Sterilin standard 55 mm petri dishes</td><td>ThermoFisher</td><td>P55V</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-02</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>50 ml polypropylen conical tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 R</td><td></td></tr><tr><td>Normal goat serum (NGS)</td><td>Cell Signalling</td><td>5425S</td><td></td></tr><tr><td>35 &amp;mu;m cell strainer cap</td><td>BD</td><td>352235</td><td></td></tr></tbody></table>

brain cell isolation from zebrafish larvae: a technique to obtain intact neurons, macrophages, and microglia from zebrafish brain tissue

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

Source: Mazzolini, J. et al. <a href='https://www.jove.com/v/57431/isolation-rna-extraction-neurons-macrophages-microglia-from-larval'>Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains.</a> J. Vis. Exp. (2018)
This video describes a protocol to isolate neurons, macrophages, and microglia from larval zebrafish brains by mechanically disrupting the brain tissue. The purified cell populations can further be used for genomic analysis.

Source: Mazzolini, J. et al. Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains. J. Vis. Exp. (2018)
This ...

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"&#160;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.

brain-cell-isolation-from-zebrafish-larvae-technique-to-obtain-intact

Research

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

Ex vivo studies

4.1K Views. 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...

Abstract

Protocol

Disclosures

<table><tbody><tr><td>1-phenyl 2-thiourea (PTU)</td><td>Sigma</td><td>P7629</td><td></td></tr><tr><td>Hepes</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G8644-100ML</td><td></td></tr><tr><td>HBSS 1X</td><td>Gibco</td><td>14170-088</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-02</td><td></td></tr><tr><td>HBSS 10X</td><td>Gibco</td><td>14180-046</td><td></td></tr><tr><td>DPBS 1X</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Tricaine (MS222)</td><td>Sigma</td><td>A5040</td><td></td></tr><tr><td>Sterilin standard 90 mm petri dishes</td><td>ThermoFisher</td><td>101VIRR</td><td></td></tr><tr><td>Surgical micro-scissors</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>3 mL Pasteur plastic bulk pipette</td><td>SLS</td><td>PIP4206</td><td></td></tr><tr><td>Glass homogenizer</td><td>Wheaton</td><td>357538</td><td></td></tr><tr><td>Sterilin standard 55 mm petri dishes</td><td>ThermoFisher</td><td>P55V</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-02</td><td></td></tr><tr><td>40 &micro;m cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>50 ml polypropylen conical tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 R</td><td></td></tr><tr><td>Normal goat serum (NGS)</td><td>Cell Signalling</td><td>5425S</td><td></td></tr><tr><td>35 &mu;m cell strainer cap</td><td>BD</td><td>352235</td><td></td></tr></tbody></table>

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

In This Article

Abstract

Protocol

Disclosures

Materials

Reprints and Permissions

Tags

Related Articles

Name	Company	Catalog Number
1-phenyl 2-thiourea (PTU)	Sigma	P7629
Hepes	Gibco	15630-056
D-Glucose	Sigma	G8644-100ML
HBSS 1X	Gibco	14170-088
Percoll	GE Healthcare	17-0891-02
HBSS 10X	Gibco	14180-046
DPBS 1X	Gibco	14190-094
Tricaine (MS222)	Sigma	A5040
Sterilin standard 90 mm petri dishes	ThermoFisher	101VIRR
Surgical micro-scissors	Fine Science Tools	15000-00
3 mL Pasteur plastic bulk pipette	SLS	PIP4206
Glass homogenizer	Wheaton	357538
Sterilin standard 55 mm petri dishes	ThermoFisher	P55V
Percoll	GE Healthcare	17-0891-02
40 µm cell strainer	Falcon	352340
50 ml polypropylen conical tube	Falcon	352070
Centrifuge	Eppendorf	5804 R
Normal goat serum (NGS)	Cell Signalling	5425S
35 μm cell strainer cap	BD	352235