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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. Solution and Media Preparation
<ol>
	<li>Prepare stock and buffer solutions as per Table 1. Prepare working solutions as per Table 2.</li>
</ol>
2. Culture and Maintenance of F9 RARE-LacZ Cells
<ol>
	<li>Thawing Frozen Cells&#8203;
	<ol>
		<li>Prepare F9 RA response element (RARE)-LacZ cell culture medium as detailed in Table 2.</li>
		<li>Coat 10 cm culture dishes (cell culture treated) with 10 ml of 0.2% gelatin (refer to Table 2) for 30 min at RT. Wash off excess gelatin with two rinses of 10 ml distilled water. Immediately add 9 ml of F9 RARE-LacZ cell culture medium into the flask to prevent the gelatin from drying.</li>
		<li>Thaw a vial of F9 RARE-LacZ cells in a 37 &#176;C water bath for 2 - 3 min. Plate cells (around 1 x 106 cells/ml) into the coated dish containing 9 ml of culture medium. Culture at 37 &#176;C, 5% CO2. Change the medium the next day.</li>
	</ol>
	</li>
	<li>Maintenance of F9 RARE-LacZ Cells
	<ol>
		<li>Maintain a regular passage every 2 - 3 days at a ratio of 1:10. 
		Note: Do not let cells grow into confluency as this will result in their differentiation.</li>
		<li>Split the cultures at a confluency of 80-90%. Remove the media and wash the cells with 10 ml of 1x phosphate-buffered saline (PBS). Remove the PBS and add 1 ml of trypsin, incubate for 5 min. Add 9 ml of culture medium, pipette up and down and split cells 1:10 for passage.</li>
	</ol>
	</li>
	<li>Freezing F9 RARE-LacZ Cells
	<ol>
		<li>Trypsinize a 10 cm dish containing F9 RARE-LacZ cells at 80 - 90% confluence as detailed in step 2.2.2. Transfer the dissociated cells into a 15 ml centrifuge tube and spin at 200 x g for 5 min.</li>
		<li>Remove supernatant and resuspend cell pellet in 10 ml of F9 RARE-LacZ freezing medium (see Table 2). Aliquot 1 ml into labeled cryovials and transfer vials into freezing containers. Place freezing container in -80 &#176;C freezer overnight (O/N) and transfer vials to liquid nitrogen tanks the next day.</li>
	</ol>
	</li>
</ol>
3. Dissection of Embryonic Day 8.5 (E8.5) Embryos
<ol>
	<li>Mating Cage Set-up and Plug Check&#8203;
	<ol>
		<li>Set-up a mating cage containing one mating pair. The morning of the next day, check for the presence of a vaginal plug and designate the day a plug is found as day 0.5. At day E8.5, harvest embryos.</li>
	</ol>
	</li>
	<li>Dissection of Embryos
	<ol>
		<li>Sacrifice the dam through cervical dislocation and spray the abdominal area with 70% ethanol (see Table 2). Make a transverse cut on the skin above the abdomen using surgical scissors and pull the two flaps apart (anterior and posterior) to expose the abdominal area. Make a similar cut on the peritoneum to expose the abdomen.</li>
		<li>Locate the uteri and release uteri from the oviducts and mesometrium using a pair of scissors. Place the uteri in a 6 cm dish (non-cell culture treated) with 10 ml 1x PBS to wash off excess fat and blood. Transfer to another 6 cm dish containing fresh 10 ml 1x PBS.</li>
		<li>Separate one uterus containing one embryo by cutting it off using surgical scissors. Remove the uterus and decidua and release the yolk sac containing the embryo using two pairs of no. 5 forceps.</li>
		<li>Separate the yolk sac from the embryo and transfer the yolk sac to a 1.5 ml microcentrifuge tube for genomic DNA extraction to be used for genotyping. Use a P20 pipette with a wide-bore tip, transfer the whole embryo into a 1.5 ml microcentrifuge tube containing 10 &#181;l of trypsin. Place the tube on ice.</li>
		<li>Repeat steps 3.2.3 - 3.2.4 until all embryos have been dissected.</li>
	</ol>
	</li>
	<li>Embryo Dissociation
	<ol>
		<li>Incubate the 1.5 ml tubes containing embryos in trypsin at 37 &#176;C for 5 min to facilitate enzymatic dissociation.
		<ol>
			<li>&#160;Triturate gently using a P20 pipettor for mechanical dissociation of the embryo, and plate the whole 10 &#181;l volume containing one dissociated embryo over one well of F9 RARE-LacZ cells in a 96-well plate (see step 4.1 for plating F9 RARE-LacZ cells in a 96-well plate). Culture O/N at 37 &#176;C and 5% CO2.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. RA Assay of E8.5 Embryos
<ol>
	<li>Plating F9 RARE-LacZ in 96-well plate&#8203;
	<ol>
		<li>One day prior to harvesting E8.5 embryos or dissociation of neurospheres, coat a 96-well plate with 100 &#181;l of 0.2% gelatin per well for 30 min at RT. Aspirate out the gelatin and rinse twice with 200 &#181;l of distilled water.</li>
		<li>Trypsinize F9 RARE-LacZ cells maintained in 10 cm dishes as detailed in step 2.2.2. Plate 1 x 105 cells/well of F9-RARE LacZ cells, but this time using culture medium without G418.</li>
		<li>Prepare enough wells for co-culture of embryos (~ 12 - 20, depending on litter size), neurospheres (3 wells/genotype) as well as for a standard curve in triplicate (27 wells).</li>
	</ol>
	</li>
	<li value="2">Co-culture of F9 RARE-LacZ and E8.5 Embryos
	<ol>
		<li>Harvest embryos as detailed in section 3.2 and plate on top of F9 RARE-LacZ in the 96-well plate from section 4.1. Culture O/N at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
</ol>
Table 1. Buffers and Solution Compositions.
<table>
	<tbody>
		<tr>
			<td>Stock Buffers/Solutions</td>
			<td>Components</td>
			<td>Storage conditions</td>
		</tr>
		<tr>
			<td>10 mM (3 mg/ml) all-trans retinoic acid</td>
			<td>Dissolve 50 mg in 16.67 ml absolute ethanol. Prepare 1 ml aliquots.</td>
			<td>-80 &#186;C in the dark, in 1.5 ml amber microcentrifuge tubes</td>
		</tr>
	</tbody>
</table>
Table 2. Working Buffers and Solution Compositions.
<table>
	<tbody>
		<tr>
			<td>Working Buffers/Solutions</td>
			<td>Components</td>
			<td>Storage conditions</td>
		</tr>
		<tr>
			<td>F9 RARE-LacZ cell culture medium</td>
			<td>50 ml fetal bovine serum (FBS) (final concentration 10%)</td>
			<td>Filter-sterilize.</td>
		</tr>
		<tr>
			<td></td>
			<td>5 ml 100x Penicillin Streptomycin (final conc 1X)</td>
			<td>Store at 4 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td>4 ml 50 mg/ml Geneticin (G418, final conc 0.4 mg/ml)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>to 500 ml Dulbecco&#39;s modified Eagle medium/nutrient mixture F-12 (DMEM/F-12, with L-glutamine and 4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid [HEPES])</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2% gelatin</td>
			<td>25 ml 2.0% gelatin</td>
			<td>Store at 4 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td>80 ml distilled water</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Mix well</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>30 ml 95% ethanol</td>
			<td>Place in spray bottle.</td>
		</tr>
		<tr>
			<td></td>
			<td>70 ml distilled water</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>F9 RARE-LacZ freezing medium</td>
			<td>F9 RARE-LacZ culture medium + 5% DMSO</td>
			<td>prepare fresh every time</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C3H/HeSn-Gpr161vl/J mouse strain</td>
			<td>Jackson Laboratory</td>
			<td>316</td>
			<td></td>
		</tr>
		<tr>
			<td>F9 RARE-LacZ reporter cell line</td>
			<td></td>
			<td></td>
			<td>from Dr. Michael Wagner (SUNY Downstate Medical Center) and Ben Thiede (Dr. Jeff Corwin lab, University of Virginia)</td>
		</tr>
		<tr>
			<td>DMEM/F-12 (with L-glutamine and HEPES)</td>
			<td>ThermoFisher Scientific</td>
			<td>11330-057</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), qualified</td>
			<td>ThermoFisher Scientific</td>
			<td>26140-079</td>
			<td>Store at -20 &#176;C in 10-ml aliquots to avoid repeated freeze-thaw; warm in 37 C for media preparation</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140-122</td>
			<td>Store at -20 &#176;C in 10-ml aliquots to avoid repeated freeze-thaw; light-sensitive</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>ThermoFisher Scientific</td>
			<td>10131-027</td>
			<td>Store at 4 &#176;C; light-sensitive</td>
		</tr>
		<tr>
			<td>2% Gelatin</td>
			<td>Sigma Aldrich</td>
			<td>G1393</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>41639</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>all-trans Retinoic Acid (at-RA)</td>
			<td>Sigma Aldrich</td>
			<td>R-2625</td>
			<td>Store 1 ml aliquots of stock in -80 &#176;C; extremely sensitive to light</td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>ThermoFisher Scientific</td>
			<td>12605-010</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>25200-056</td>
			<td>alternative to TrypLE Express; aliquot and store at -20 C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>ThermoFisher Scientific</td>
			<td>14170-112</td>
			<td>Store at 4 C</td>
		</tr>
		<tr>
			<td>10X phosphate buffered solution (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>10010-023</td>
			<td>Store at 4 C</td>
		</tr>
		<tr>
			<td>95% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing container</td>
			<td>ThermoFisher Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish (non-cell culture treated)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish (cell culture treated)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm culture dish (non-cell culture treated)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well cell culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml amber microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml cryovials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>surgical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fine surgical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>no. 5 forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>blunt-ended forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel blade</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass pasteur pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>alcohol lamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

employing reporter cells in a co-culture to study retinoic acid production by mouse embryonic cells

Source: Ababon, M. R., et al. <a href="https://app.jove.com/v/54443">Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for assessing retinoic acid activity in mouse embryos. Upon isolating cells from the mouse embryos, the cells are co-cultured with retinoic acid (RA) reporter cells. Embryonic cells produce RA, which induces beta-galactosidase expression in reporter cells, indicating RA activity in the embryonic cells.

Employing Reporter Cells in a Co-Culture to Study Retinoic Acid Production by Mouse Embryonic Cells

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. Solution and Media Preparation

	...

Take a mouse embryo at an early developmental stage immersed in a buffer. Transfer the embryo into an enzyme solution and incubate it under physiological conditions, allowing the enzymes to break down the intercellular connections.
Mechanically dissociate the digested embryo to form a cell suspension.
Transfer the embryonic cells onto an adherent culture of reporter cells.
The reporter cells carry a transgenic construct containing the lacZ gene, which encodes the enzyme beta-galactosidase. The expression of lacZ is regulated by a retinoic acid response element, or RARE.
The embryonic cells release retinoic acid, or RA, a signaling molecule for embryonic development.
RA enters the reporter cells and binds to its receptor complex on the RARE, triggering the formation of a transcriptional activator complex.
The complex activates the expression of lacZ, resulting in the production of beta-galactosidase.
Beta-galactosidase production in the reporter cells indicates the release of RA by the embryonic cells.

employing-reporter-cells-co-culture-to-study-retinoic-acid-production

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Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

27 Views. Source: Ababon, M. R., et al. Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay. J. Vis. Exp. (2016). This video demonstrates a technique for assessing retinoic acid activity in mouse embryos. Upon isolating cells from the mouse embryos, the cells are co-cultured with retinoic acid (RA) reporter cells. Embryonic cells produce RA, which induces beta-galactosidase expression in reporter cells, indica...

Video: Employing Reporter Cells in a Co-Culture to Study Retinoic Acid Production by Mouse Embryonic Cells - Concept

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

JoVE Journal

Developmental Biology

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

MATLAB Optimization for Analyzing Mitochondrial Networks in Neuroscience

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in timelapse files, considerably simplifying and speeding up the process of image processing. Nonetheless, existing tools produce a large output volume, requiring individual manual attention, and basic experimental setups have an output of thousands of files, each requiring extensive and time-consuming handling. 
To address these issues, a routine optimization was developed, in both MATLAB code and live-script forms, allowing for swift file analysis and significantly reducing document reading and data processing. With a speed of 100 files/min, the optimization allows an overall rapid analysis. The optimization achieves the results output by averaging frame-specific data for individual mitochondria throughout time frames, analyzing data in a defined manner, consistent with those output from existing tools. Live confocal imaging was performed using the dye tetramethylrhodamine methyl ester, and the routine optimization was validated by treating neuronal cells with retinoic acid receptor (RAR) agonists, whose effects on neuronal mitochondria are established in the literature. The results were consistent with the literature and allowed further characterization of mitochondrial network behavior in response to isoform-specific RAR modulation. 
This new methodology allowed rapid and validated characterization of whole-neuron mitochondria network, but it also allows for differentiation between axon and cell body mitochondria, an essential feature to apply in the neuroscience field. Moreover, this protocol can be applied to experiments using fast-acting treatments, allowing the imaging of the same cells before and after treatments, transcending the field of neuroscience.

Author Spotlight: An Optimized Automated Method for Investigating Retinoic Acid Receptors in Neuronal Mitochondria

The mitochondrial network is extremely complex, making it very challenging to analyze. A novel MATLAB tool analyzes live confocal imaged mitochondria in timelapse images but results in a large output volume requiring individual manual attention. To address this issue, a routine optimization was developed, allowing for speedy file analysis.

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in ...

Employing Reporter Cells in a Co-Culture to Study Retinoic Acid Production by Mouse Embryonic Cells

Transcript

Employing Reporter Cells in a Co-Culture to Study Retinoic Acid Production by Mouse Embryonic Cells

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors