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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&#39;&#39;.</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&#39;&#39;&#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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;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 &#956;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

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.

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

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.

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1.4K Views. Source: Mazzolini, J. et al. Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains. 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. 

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

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

JoVE Journal

Neuroscience

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsr&#248;d et al. designed for rat liver cell isolation and provides high yield (up to 14 x 106 cells per mouse) and high purity (&#62;95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient.

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle ...

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

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