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1. Preparation of solutions
<ol>
	<li>E2 media (1x)
	<ol>
		<li>Prepare 100x E2A (500 mL), 500x E2B (100 mL) and 500x E2C (100 mL) solutions by combining all components shown in Table 1. Autoclave E2A, E2B and E2C solutions. Store at 4 &#176;C.</li>
		<li>For 1x E2 media: Combine 5 mL of 100x E2A, 1 mL of 500x E2B, and 1 mL of 500x E2C. Bring the volume to 500 mL with sterile water. Adjust the pH to 7.0-7.5.</li>
		<li>Prepare 50 mL aliquots of 1x E2 media and store at -20 &#176;C for long-term storage. However, precipitations can occur upon thawing. Make sure precipitation is fully dissolved before using the stock solution.</li>
	</ol>
	</li>
</ol>
Table 1: Components of 1x E2 media for zebrafish cell culture.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Component</td>
			<td>Amount</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>100X E2A (500mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>NaCl</td>
			<td>43.8 g</td>
			<td>1500 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>KCl</td>
			<td>1.88 g</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>MgSO4</td>
			<td>6 g</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>KH2PO4</td>
			<td>1.03 g</td>
			<td>15 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>Na2HPO4</td>
			<td>0.34 g</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>500X E2B (100 mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>CaCl2</td>
			<td>5.5 g</td>
			<td>500 mM</td>
		</tr>
		<tr>
			<td>500X E2C (100 mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>NaHCO3</td>
			<td>3 g</td>
			<td>350 mM</td>
		</tr>
		<tr>
			<td>1X E2 (500 mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100X E2A</td>
			<td>5 mL</td>
			<td>1X</td>
		</tr>
		<tr>
			<td></td>
			<td>500X E2B</td>
			<td>1 mL</td>
			<td>1X</td>
		</tr>
		<tr>
			<td></td>
			<td>500X E2C</td>
			<td>1 mL</td>
			<td>1X</td>
		</tr>
	</tbody>
</table>
<ol start="2">
	<li>E3 Media (1x)
	<ol>
		<li>Dissolve the components in 1 L sterile water as shown in Table 2 to make 100x stock. Dilute the stock in sterile water to make 1x E3 media.</li>
		<li>Add 0.2% methylene blue. For 20 mL of 1x E3 media, add 40 &#181;L of methylene blue.</li>
	</ol>
	</li>
</ol>
Table 2: Components of 100x E3 media for maintaining zebrafish embryos.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Amount (g)</td>
			<td>Concentration in 100X stock (mM)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>29.22</td>
			<td>500</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>1.26</td>
			<td>17</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>4.85</td>
			<td>33</td>
		</tr>
		<tr>
			<td>MgSO4 7H2O</td>
			<td>8.13</td>
			<td>33</td>
		</tr>
	</tbody>
</table>
<ol start="3">
	<li>80x saline stock solution
	<ol>
		<li>Combine all components shown in Table 3. Add water to make 100 mL solution. Mix until all components are dissolved. Store the solution at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
Table 3: Components of 80x saline solution for zebrafish cell culture media.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Amount (g)</td>
			<td>Concentration in stock (mM)</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>1.44</td>
			<td>80</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>0.44</td>
			<td>40</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>0.148</td>
			<td>10</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>6.1</td>
			<td>256</td>
		</tr>
	</tbody>
</table>
<ol start="4">
	<li>Zebrafish cell culture medium (ZFCM+)
	<ol>
		<li>Combine all components shown in Table 4 to make 250 mL media. Adjust the pH to 7.5. Filter media using 0.22 &#181;m filter and store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
Table 4: Components of zebrafish cell culture medium with serum and antibiotics.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Amount (mL)</td>
			<td>Volume %</td>
		</tr>
		<tr>
			<td>L-15 medium (with phenol red)</td>
			<td>212.75</td>
			<td>85.1</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>5</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>1</td>
			<td>0.4</td>
		</tr>
		<tr>
			<td>80X Saline solution</td>
			<td>3.125</td>
			<td>1.25</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>28.125</td>
			<td>11.25</td>
		</tr>
	</tbody>
</table>
2. Primary retinal ganglion cell culture derived from zebrafish embryos
NOTE: Perform steps 2.1 and 2.2 in a laminar flow hood.
<ol>
	<li>Preparation of coverslips
	<ol>
		<li>Prepare 4-6 culture plates in each experiment. Use acid-cleaned coverslips (22 x 22 mm square; 0.16-0.19 mm thickness) that are stored in 100% ethanol.</li>
		<li>Remove one coverslip from the storage container by using forceps and flame it to remove residual ethanol.</li>
		<li>Air dry the coverslip completely by placing it at an angle inside a 35-mm culture dish.</li>
		<li>Prepare Poly-D-Lysine (PDL) working solution (1x) by diluting 10x stock (5 mg/mL) in sterile water.</li>
		<li>Apply 100 &#181;L of 0.5 mg/mL PDL to the center of each coverslip and avoid spreading of the solution to the edges.</li>
		<li>Incubate the PDL on coverslips for 20-30 min at room temperature (RT). Make sure the PDL does not dry out.</li>
		<li>Wash the PDL with 0.5 mL sterile water three times. Let the plates dry completely.</li>
		<li>Prepare laminin working solution (1x) by diluting 50x stock (1 mg/mL) in 1x PBS.</li>
		<li>Apply 100 &#181;L of 20 &#181;g/mL laminin in PBS to the center of each coverslip and avoid the spread of solution to the edges.</li>
		<li>Incubate the plates at 37 &#176;C in a humidified incubator for 2-6 h. Avoid drying of the laminin solution.</li>
	</ol>
	</li>
	<li>Embryo dissection and plating RGCs
	<ol>
		<li>Prepare and label four 35-mm tissue culture dishes and fill with 4 mL of: 70% ethanol, &#34;E2 media 1&#34;, &#34;E2 media 2&#34;, &#34;E2 media 3&#34; on the day of dissection. Keep the dishes in the fridge until dissections.</li>
		<li>When zebrafish embryos are 34 hours post fertilization (hpf), take the culture dishes coated with laminin out of the incubator and wash the coverslips three times with 0.5 mL of 1x PBS.</li>
		<li>After the final wash, add 4 mL of ZFCM(+) media to each culture dish and avoid drying the plate.</li>
		<li>Retrieve the prepared culture dishes from Step 2.2.1. Let them equilibrate to RT.</li>
		<li>Fill 4-6 PCR tubes with 15 &#181;L of ZFCM(+) media. One tube is needed to prepare RGCs from 4 eyes to be plated onto one coverslip.</li>
		<li>Retrieve zebrafish embryos from the incubator and immerse embryos in 35 mm tissue culture dish containing 70% ethanol for 5-10 s to sterilize.</li>
		<li>Using a transfer pipette, transfer embryos to E2 Media 1 dish containing sterile E2 media to wash excess ethanol.</li>
		<li>Transfer embryos to E2 Media 2 dish and remove their chorions with sharp forceps.</li>
		<li>Transfer embryos to final E2 Media 3 dish to perform dissections.</li>
		<li>Using a pair of fine forceps, dissect out the retinas.</li>
		<li>Position and hold embryos anterior to their yolk with one of the forceps and remove the tail posterior to the yolk sac with the other forceps.</li>
		<li>Grab the neck with forceps and take off the head to expose brain and eyes to the E2 media. Avoid cutting the yolk sac.</li>
		<li>With the tip of fine forceps, gently roll the eyes off from the head, while holding the cranial tissue down with the second forceps. Keep eyes isolated from the adjacent tissue debris.</li>
		<li>Transfer four eyes to one of the previously-prepared tubes containing ZFCM(+).</li>
		<li>Gently titrate up and down with the P20 pipette and a yellow tip about 45 times to dissociate cells. Avoid any air bubbles.</li>
		<li>Transfer the ZFCM(+) with dissociated cells to the center of the coverslip. Repeat steps 10-12 for additional coverslips.</li>
		<li>Maintain cultures on benchtop at 22 &#176;C on a polystyrene foam rack to absorb vibrations.</li>
		<li>Perform imaging 6-24 h after plating.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35-mm culture dish</td>
			<td>Sarstedt</td>
			<td>83-3900</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Corning</td>
			<td>2850-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes (100 x 15 mm)</td>
			<td>VWR</td>
			<td>25384-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26140087</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>ThermoFisher Scientific</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium without phenol red</td>
			<td>Gibco/Fisher Scientific</td>
			<td>21-083-027</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone/Fisher Scientific</td>
			<td>SH3025601</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine (PDL)</td>
			<td>Sigma Aldich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma Aldich</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Steritop 0.22 &#956;m filter</td>
			<td>&#160;Millipore&#160;</td>
			<td>S2GPT05RE</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of primary retinal ganglion cell cultures from zebrafish embryos

Source: Terzi, A., et al., <a href="https://app.jove.com/v/62165">ROS Live Cell Imaging During Neuronal Development.</a> J. Vis. Exp. (2021)
The video demonstrates a method for culturing primary retinal ganglion cells from zebrafish embryos. Early-stage embryos undergo dissection to isolate the eyes, which are subsequently mechanically dissociated into a suspension of retinal ganglion cells. This cell suspension is cultured on coverslips coated with adhesive proteins to facilitate axon development.

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

1. Preparation of solutions

	E2 media (1x)
	
		Prepare 100x E2A (500 mL), 500x E2B (100 mL) and 500x E2C (100 mL) solutions by combining all components ...

Begin with early developmental stage zebrafish embryos in embryo media.
Remove the chorion, the protective membrane surrounding the embryos, and transfer the embryos to a fresh culture plate containing media.
Remove the tail portion of the embryo, then separate the head and gently detach the eyes from the head.
The eyes contain the retina, a thin layer of tissue which houses the retinal ganglion cells.&#160;
Transfer the eyes to a tube containing zebrafish cell culture media.
Mechanically dissociate the eyes to release the retinal ganglion cells and form a single-cell suspension.
Take coverslips placed in a culture plate containing zebrafish culture media.
The coverslips are coated with a synthetic polymer and an adhesion protein.
Transfer the cells onto the center of the coverslip and incubate.
Retinal ganglion cells attach to the coated surfaces, and further, these cells develop long projections called axons.

generation-primary-retinal-ganglion-cell-cultures-from-zebrafish

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

40 vistas. Fuente: Terzi, A., et al., ROS Imágenes de células vivas durante el desarrollo neuronal. J. Vis. Exp. (2021) El video muestra un método para cultivar células ganglionares primarias de la retina de embriones de pez cebra. Los embriones en etapa temprana se someten a disección para aislar los ojos, que posteriormente se disocian mecánicamente en una suspensión de células ganglionares de la retina. Esta suspensión celular se cultiva en cubreobjetos recubiertos con proteínas adhesivas par...

Video: Generación de cultivos primarios de ganglios angulares de la retina a partir de embriones de pez cebra - Concepto

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Cultura de adulto pez cebra transgénico de retina explantes de Células vivas imágenes de microscopía multifotónica

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Investigación

JoVE Journal

Biología del Desarrollo

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

Cultura y transfección de células primarias de pez cebra

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

Preparación frescas rebanadas retinales de pez cebra adulto para Ex Vivo de experimentos

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

Transcripción

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos