We would like to acknowledge Dr. Michael Wagner (SUNY Downstate Medical Center) and Ben Thiede (Dr. Jeff Corwin lab, University of Virginia) for providing us with the F9 RARE-LacZ cells and technical support. This study is funded by the New Jersey Commission on Spinal Cord Research (NJCSCR) (Grantnumber:10-3092-SCR-E-0) and the New Jersey Commission on Brain Injury Research (NJCBIR) (Grant number: CBIR13FEL006).

All animal work was done according to approved Rutgers-RWJMS IACUC methods.
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
	<ol>
		<li>Prepare F9 RARE-LacZ cell culture medium as detailed in Table 2.</li>
		<li>Coat 10 cm culture dishes (cell culture treated) with 10 ml&#160; 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 cells14 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 O/N and transfer vials to liquid nitrogen tanks the next day.</li>
	</ol>
	</li>
</ol>
3. Dissection of E8.5 Embryos
<ol>
	<li>Mating Cage Set-up and Plug Check
	<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 0.523. 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 scissors23. 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 forceps23.</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>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 5.1 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. Laminectomy, Dissection of Adult Lumbar Spinal Cord and Neurosphere Culture
<ol>
	<li>Laminectomy
	<ol>
		<li>Sacrifice an adult (P60) mouse through cervical dislocation.</li>
		<li>Spray the dorsal side with 70% ethanol and make a transverse cut using surgical scissors. Pull both flaps (anterior and posterior) apart to expose the back and vertebral column.</li>
		<li>Using surgical scissors, trim off the muscles covering the spine. Locate the lumbar region of the spinal cord, below the ribs. Using the scissors, make a deep cut to sever the spine at this point to expose the spinal cord.</li>
		<li>Pull up the lumbar region of the spine with no. 5 forceps so that the exposed transverse section of the spinal cord is facing the experimenter. Using the tips of fine scissors, snip the vertebra and muscle at the 3 o&#39;clock and 9 o&#39;clock positions of the exposed end. Grasp the dorsal flap of the cut vertebra with forceps. Continue these cuts caudally until the length of the lumbar spinal cord is exposed.</li>
		<li>Release the spinal cord from the vertebrae using no. 5 as well as blunt-ended forceps. Transfer the spinal cord to a 15 ml tube containing 3 ml of DMEM/F12 culture medium. Keep on ice until all lumbar spinal cords have been isolated.</li>
	</ol>
	</li>
	<li>Spinal Cord Dissection
	<ol>
		<li>Pour the culture medium with the spinal cord into a 6 cm dish (non-cell culture treated).</li>
		<li>Under a stereomicroscope, remove the dorsal root ganglia and blood vessels around the spinal cord segment by grasping the ganglia and blood vessels using two pairs of no. 5 forceps and gently pulling them away from the spinal cord. Transfer the spinal cord segment to a 6 cm dish with no medium and mince the tissue finely using a scalpel blade.</li>
		<li>Transfer minced tissue to 15 ml tube containing 1 ml of neurosphere dissociation media (see Table 2). Put tube on ice and repeat steps 4.2.1 - 4.2.2 until all spinal cords have been processed.</li>
		<li>Incubate tubes containing minced tissue in dissociation medium at 37 &#176;C for 10 min, flick the tube on the side to mix and then incubate at 37 &#176;C for another 10 min. After enzymatic digestion, triturate with fire-polished pipettes of decreasing diameters for mechanical dissociation. 
		Note: Prepare fire-polished pipettes of decreasing diameters prior to performing experiments. Take a glass Pasteur pipette and plug the wide end with clean cotton. Using an alcohol lamp, fire polish the tips of the pipettes in the flame to create different diameters: &#34;regular&#34;, &#34;fine&#34;, and &#34;extra fine&#34;. For &#34;regular&#34; polished pipettes, twirl the end of the pipette in the flame to round of the edges without decreasing the diameter (~ 1 mm). Rotate the end of the pipette in the flame for a slightly longer periods to create &#34;fine&#34; (~ &#62;1 mm and &#62; 5 mm diameter) and &#34;extra fine&#34; (~ 0.5 mm) polished pipettes. Do not use pipettes with diameters less than 0.5 mm.</li>
	</ol>
	</li>
	<li>Neurosphere Culture
	<ol>
		<li>Spin down tubes containing dissociated spinal cord tissue at 200 x g for 10 min. Remove supernatant and resuspend cell pellet in 1 ml of neurosphere culture media (see Table 2). Triturate with a P1,000 pipette followed by a P200 pipette to facilitate dissociation. 
		Note: Avoid introducing bubbles, which would decrease cell viability.</li>
		<li>Transfer 10 &#181;l of dissociated cells into a hemocytometer and obtain a cell count. Plate cells at a density of 1x105/10 ml in a T25 flask with 10 ml of neurosphere culture media. Incubate at 37 &#176;C and 5% CO2 for 7 days until neurospheres appear 24.</li>
	</ol>
	</li>
	<li>Neurosphere Dissociation
	<ol>
		<li>Collect neurosphere culture and transfer to 15 mL tubes. Spin at 200 x g for 10 min. Remove most of the supernatant leaving 1 ml behind. Triturate with a P1,000 pipette to resuspend the cell pellet and dissociate neurospheres into single cells. Transfer 10 &#181;l of dissociated cells into a hemocytometer and get a cell count.</li>
		<li>Plate 1 x 105 dissociated neurosphere cells over one well of F9 RARE-LacZ in a 96-well plate (see section 5.1 for plating F9 RARE-LacZ in 96-well plate). Plate 3 wells as replicates. Culture O/N at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
</ol>
5. RA Assay of E8.5 Embryos and Spinal Cord Neurospheres
<ol>
	<li>Plating F9 RARE-LacZ in 96-well plate
	<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>Co-culture of F9 RARE-LacZ and E8.5 Embryos or Spinal Cord Neurospheres
	<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 5.1. In a similar manner, dissociate neurospheres and plate on top of F9 RARE-LacZ in the 96-well plate as detailed in step 4.4. Culture O/N at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Generating a Standard Curve
	<ol>
		<li>To generate a standard curve, prepare all-trans RA (at-RA) solutions with the following concentrations: 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, 100 fM, by serial dilution of the at-RA stock (see Table 1) with F9 RARE-LacZ culture medium.</li>
		<li>Add 100 &#181;l of these different at-RA concentrations to F9 RARE-LacZ cells from section 5.1. Assign wells containing untreated F9 RARE-LacZ cells as negative control. Prepare triplicates of each RA concentration and negative control wells. Culture O/N at 37 &#176;C and 5% CO2. 
		Note: F9 RARE-LacZ cells respond in a dose-dependent linear manner to varying concentrations of all-trans RA.</li>
	</ol>
	</li>
	<li>RA Assay
	<ol>
		<li>After O/N culture of the 96-well plate containing the standard curves and co-cultures, remove the media and fix the cultures by adding 100 &#181;l of 2.5% glutaraldehyde (see Table 2) for 15 min, RT. Remove the fixative and wash twice with 200 &#181;l 1x&#160; PBS, 10 min per wash.</li>
		<li>Remove PBS and wash three times with 200 &#181;l of the LacZ wash solution (see Table 2), 10 min per wash. During the third wash, prepare the X-gal staining solution in a 15 ml tube wrapped in foil (see Table 2). Calculate how much staining solution is needed and prepare the appropriate amount (200 &#181;l/well). 
		Note: X-gal staining solution is light sensitive. Use the staining solution within 15 min after preparation.</li>
		<li>Prepare a humidified chamber by placing a paper towel dampened with distilled water into a plastic container big enough to hold a 96-well plate. Add 200 &#181;l of the X-gal staining solution per well of the 96-well plate and place the plate in a humidified chamber.</li>
		<li>Incubate humidified chamber containing the 96-well plate at 37 &#176;C to allow for development of blue-colored precipitate. For E8.5 embryos, incubate the plate O/N (12 - 16 hr). For neurosphere cultures, incubate the plate for 4 - 8 hr.</li>
	</ol>
	</li>
	<li>Reading Absorbance at OD610 and Imaging
	<ol>
		<li>After color reaction, remove the LacZ staining solution and replace with 200 &#181;l 1x PBS. Measure the absorbance at 610 nm using a standard ELISA plate reader to quantify the reporter response to RA. Image individual wells using a standard stereomicroscope equipped with a camera.</li>
	</ol>
	</li>
</ol>
6. Subcloning of F9 RARE-LacZ Cells
<ol>
	<li>Checking F9 RARE-LacZ rResponse to RA
	<ol>
		<li>Before starting any experiments, test the response of F9 RARE-LacZ cells to RA. Plate F9 RARE-LacZ cells in 96-well plate (see section 5.1), and add 1 nM all-trans retinoic acid (see step 5.3). Incubate in a humidified chamber at 37 &#176;C O/N.</li>
		<li>Visualize development of blue colored precipitate using a microscope. If color development is not uniform throughout the cells in the well (see Figure 1B, left), perform subcloning detailed below.</li>
	</ol>
	</li>
	<li>Subcloning of F9 RARE-LacZ Cells
	<ol>
		<li>Split the F9 RARE-LacZ cells cultured in 10 cm dishes as detailed in step 2.2.2. Instead of a 1:10 ratio, plate a low seeding density of 1 x 105cells/10 ml in a 10 cm dish to allow for growth of isolated colonies such that each colony arises from a single F9 RARE-LacZ cell.</li>
		<li>After two days, coat a 24-well plate with 500 &#181;l of 0.2% gelatin per well for 30 min. Remove gelatin solution and rinse wells twice with 500 &#181;l distilled water. Replace water with 500 &#181;l of F9 RARE-LacZ cell culture medium.</li>
		<li>Pick a colony using a sterile P10 pipette tip and transfer into one well; do this 24 times to obtain 24 individual colonies. Culture at 37 &#176;C, 5% CO2 for 3 - 4 days.</li>
		<li>Split the individual colonies into two 24-well plates. After 2 days in culture, add 1nM of RA to each well of one 24-well plate and culture O/N. Perform LacZ staining as detailed in section 5.4 to test for high responders. Visualize color reaction of each well under a microscope to determine strong and uniform responders.</li>
		<li>Once a strong subclone responder is determined, expand the corresponding culture from the other 24-well plate. Further expand that colony in a 10 cm dish, freeze down several vials as detailed in section 2.3, and discard all other colonies. 
		Note: Only about one-fourth of the original colonies result in high responders. Typically, the first round of subcloning does not result in uniform LacZ staining and a subsequent round of subcloning needs to be done to achieve uniform strong responders.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The F9 RARE-LacZ cell line14 is a powerful system that can be used for detection of RA produced by tissue explants12,14,17,18. It is sensitive to RA concentrations as low as 100 fM, with no non-specific response detected in cells untreated with RA12,14,18. Furthermore, the reporter cells respond to all-trans RA in a linear manner within a specific range of concentration, thus allowing the LacZ reaction to be quantified and used to determine the magnitude of RA levels12,14,18. The quick and easy RA reporter assay presented here utilizes the F9 RARE-LacZ reporter cell line for visualization (Figure 2) and relative quantitative measurement (Figure 3) of embryos and cultured adult neural stem cells. This reporter system allows comparison of endogenous RA levels between individual samples, as shown in Figure 2. It is sensitive enough to be able to detect RA generated by individual E8.5 embryos (Figure 2A) as well as RA produced by cultured neurospheres (Figure 2B). The dose-dependent response of this cell line to varying concentrations of RA allows for the quantification of RA levels (Figure 3A) in the samples as well as comparison of relative RA levels between samples (Figure 3B). It is important to note that the F9 RARE reporter cell line measures the overall amount of RA generated by the tissue samples over the time of incubation. This is equivalent to the net balance of RA synthesis and catabolism.
While the F9 RARE-LacZ cell reporter system is highly sensitive and versatile, there are several critical troubleshooting steps that we have incorporated in order to ensure the reliability of this system. First, as shown in Figure 1, it is important to maintain the health of these cells by regular passages without letting them grow to confluency. Second, we have observed later passages tend to result in inconsistent RA responses, even if the cultures are constantly maintained under selection with G418. To troubleshoot this, it is recommended to discard cultures after 20 passages and thaw a new vial. In addition, regular checks must be performed to ensure that the reporter cells are still strong responders to the presence of RA in the media. Third, if the early passages display poor or non-uniform response to RA (Figure 1B, left), one or several rounds of subcloning may be necessary until strong responders are obtained (Figure 1B, right). Lastly, prior to co-culture, it is recommended that tissue samples such as embryos and neurospheres are dissociated to single cells. We have observed that dissociation of the samples results in more consistent colorimetric measurements. This may be due to a more even distribution of RA in the culture wells, as the dissociated cells are in uniform contact with the reporter cells.
Several modifications were also made to the original protocol14. First, RA response of the F9 RARE-LacZ cells were quantified by directly fixing the cells and measuring absorbance at 610 nm instead of collecting cells and analyzing lysate for b-galactosidase activity14. Another modification is the direct co-culture of dissociated samples with F9 RARE-LacZ prior to quantification of RA response. Previous methods reported culturing samples separately and adding only the media from these cultures to F9 RARE-LacZ16,18.
Despite its being a powerful system, the F9 RARE-LacZ reporter assay has several limitations. One limitation of this reporter cell line is that although it responds strongly to the presence of all-trans RA, it also responds to other retinoic acid isomers including 9-cis-RA and 13-cis-RA; however, they are 100-fold more sensitive to all-trans RA compared to the other isomers18. Another limitation to this system is that the linear dose-dependent response only falls between 10-13 M to 10-7 M at-RA14,18; thus, if comparing RA levels outside these limits, it might be necessary to perform a serial dilution of the sample until the measurements fall within the linear range. Lastly, since this is primarily a colorimetric assay, the endpoint of color development will vary between samples and experiments. Thus, it is important that a standard curve always be plated in parallel for every single experiment in order to use it for quantification of the reporter cell response.
In conclusion, we have reported the use of the F9 RARE-LacZ RA reporter cell line to measure RA levels quantitatively in E8.5 embryos and, previously unreported, in neurospheres. The versatility of this reporter system may allow the quantification of RA levels in virtually any cell or tissue type, and may result in the development of varied applications in the future.

Retinoic acid (RA) is a metabolite derivative of retinol or vitamin A and is produced through the sequential activity of several cellular dehydrogenases1. Because of its amphiphatic chemical nature, it readily crosses lipid membranes and can act as a soluble morphogenetic factor1. It is an essential molecule that regulates numerous developmental and adult cellular processes by acting as a ligand for nuclear retinoid receptors and activating gene expression2-10. Without RA, these RA receptors (RARs) bind RAREs in DNA and act as repressors; RA binding to these receptors turns them into transcriptional activators resulting in target gene expression1,5,6.
Although the RA signaling pathway is well characterized, the small size (~ 300 Da) and amphipathic nature of RA makes direct histological visualization and measurement of RA currently technically unfeasible11. The only method for direct measurement of RA levels is through isolation of RA from tissue samples using high performance liquid chromatography (HPLC)11. This method typically requires large amounts of tissue, facilitated by pooling different samples12. Thus, this technique is poorly suited for measurement of RA levels in individual embryos or small amounts of sample/tissue.
In order to investigate the cellular function of RA, several methods have been developed to indirectly infer the presence of RA. These methods make use of reporter systems containing the highly responsive RAR-beta RARE driving the expression of beta-galactosidase or luciferase11,13,14. The transgenic RARE-LacZ reporter mouse generated by Rossant et al.13 is an ideal system for visualizing spatiotemporal regions of RA signaling in situ11,13,15,16 but is unsuitable for quantitative measurements. The F9 RARE-LacZ cell line14, on the other hand, is useful for detection of RA and quantitative measurements of RA levels in tissue explants11,12,14,17,18.
F9 teratocarcinoma cells express endogenous alpha-, beta-, and gamma-retinoic acid receptors 19, and initiate differentiation into parietal endoderm after exposure to RA19-21. F9 cells have long been established as a model for RA-induced cellular differentiation, which is why it was chosen as an ideal cell line for an RA reporter assay. The F9-derived Sil-15 RA reporter cell line generated by Wagner et al.14 contains an E. coli&#39;LacZ gene downstream of one copy of the 64-bp retinoic acid response element (RARE) of the human beta-retinoic acid receptor (RAR-beta) gene. In order to select and maintain stable clones, the construct also contains the aminoglycoside phosphotransferase (NEOr) gene as a selectable marker in the presence of G418. This construct confers inducible expression of b-galactosidase in the presence of RA, which can be visualized through LacZ staining and this response can be subsequently quantified using colorimetric methods11,12,14,18.
This extremely versatile reporter cell line has been widely used in the detection of endogenous RA production through co-culture of tissue samples with the reporter cells, such as cochlear17 and embryonic floor plate explants14. In addition, this reporter line has been used for quantification of RA levels in the developing spinal cord by culturing pooled sections of embryonic spinal cords separately and adding conditioned media from these cultures to F9 RARE-LacZ cells18. Quantification was performed after LacZ staining through colorimetric reading using a standard ELISA plate reader11,12,18. Lastly, this reporter cell line has been used in the detection of the presence of RA metabolic enzymes by monitoring changes in RA levels12,18.
Here we report that the sensitivity of this reporter cell line also allows for the measurement of RA levels generated from individual co-cultured E8.5 embryos. This enables the comparison between individual embryos of different genotypes. As a specific example, Gpr161 is an orphan GPCR that regulates neurulation in part through the RA signaling pathway22, and we report using this reporter cell line to investigate the effect of a recessive mutation in Gpr161 (Gpr161vl) on overall RA levels in the embryo. In addition to this, the versatility and ease-of-use of this reporter system allows the freedom to measure and compare RA levels in different tissue samples, even in neurosphere cultures obtained from adult spinal cord stem cells. Here we describe the quantitative determination of relative RA levels in embryos and neurosphere cultures through direct co-culture with the F9 RARE-LacZ RA reporter cell line.

<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>000316</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>B-27 supplement (2 % stock solution)</td>
			<td>ThermoFisher Scientific</td>
			<td>17504-044</td>
			<td>Store at -20 &#176;C in 1-ml aliquots to avoid repeated freeze-thaw; warm at room temperature for media preparation</td>
		</tr>
		<tr>
			<td>Murine Epidermal Growth Factor (EGF)</td>
			<td>PeproTech</td>
			<td>315-09</td>
			<td>Prepare 100 &#956;g/ml stock solution in 0.1% bovine serum albumin (BSA) in 1X PBS; prepare working concentrations by diluting with 1X PBS; store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Murine Fibroblast Growth Factor (FGF)-basic</td>
			<td>PeproTech</td>
			<td>450-33</td>
			<td>Prepare 100 &#956;g/ml stock solution in 0.1% bovine serum albumin (BSA) in 1X PBS; prepare working concentrations by diluting with 1X PBS; store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>50% glutaraldehyde</td>
			<td>Amresco</td>
			<td>M155</td>
			<td>Store at 4 C</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate monohydrate (C24H39NaO4 &#183; H2O)</td>
			<td>Sigma Aldrich</td>
			<td>D5670</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>potassium ferrocyanide (K&#8324;[Fe(CN)&#8326;] &#183; 3H&#8322;O)</td>
			<td>Sigma Aldrich</td>
			<td>P-9387</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>potassium ferricyanide (K&#8323;[Fe(CN)&#8326;])</td>
			<td>Sigma Aldrich</td>
			<td>P-8131</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>X-gal (5-bromo-4-chloro-3-indolyl-&#946;-d-galactopyranoside)</td>
			<td>Promega</td>
			<td>V3941</td>
			<td>Store stock at -20 &#176;C&#160;</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>Papain</td>
			<td>Worthington</td>
			<td>LK003176</td>
			<td>Store at 4 &#176;C.&#160;</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Worthington</td>
			<td>LS003703</td>
			<td>Store in 1 ml aliquots at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Hyaluronic acid&#160;</td>
			<td>Calbiochem</td>
			<td>385908</td>
			<td>Store 200 &#956;l aliquots at -20 &#176;C.&#160;</td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>Worthington</td>
			<td>LS002139</td>
			<td>Store in 200 &#956;l aliquots at -20 C</td>
		</tr>
		<tr>
			<td>Kynurenic acid</td>
			<td>Sigma Aldrich</td>
			<td>K3375</td>
			<td>Store in 200 &#956;l aliquots at -20 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>
		<tr>
			<td>ELISA plate reader</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative measurement of relative retinoic acid levels in e8.5 embryos and neurosphere cultures using the f9 rare-lacz cell-based reporter assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

The F9 RARE-LacZ reporter is an adherent embryonic carcinoma cell line grown on gelatin-coated surface. The reporter construct allows the cells to respond quantitatively to RA with a proportional induction of beta-galactosidase. 11,12,14,18. These cells display an epithelial morphology (Figure 1A). Because of their high doubling rate, it is recommended that these cells be split every 2 - 3 days at a ratio of 1:10. During our work with this reporter cell line, we have observed that even with the addition of G418 into the culture medium, the response of these cells to RA weakens after several passages (Figure 1B, left), and this can affect the reporter capacity of this cell line. This loss of responsiveness may be due to partial loss of the transgene at later passages. As such, periodic subcloning is necessary to ensure strong and uniform responders to RA (Figure 1B, right).
While various uses of this cell line has been previously published12,14-18, reported here is the use of this cell line to measure endogenous RA levels from individual E8.5 embryos with different genotypes. The Gpr161vl/vl mutation results in decreased embryonic RA signaling22. As shown by co-culture of dissociated embryos with F9 RARE-LacZ cells, these Gpr161vl/vl embryos have reduced endogenous RA compared to wild-type littermates (Figure 2A). Because of the versatility of this reporter cell line, also reported here is a novel use of these cells to detect RA levels produced by neurosphere cultures obtained from adult Gpr161wt/wt mice (Figure 2B). This reporter cell line was able to demonstrate that neurosphere cultures from adult spinal cord stem cells produce endogenous RA. The great difference in staining intensity indicates a greater reporter cell response to spinal cord neurospheres compared to E8.5 embryos. This may be due to much greater RA levels generated by the neurospheres over time compared to E8.5 embryos, which could be due to the fact that neurosphere cultures are more homogenous than E8.5 embryos and thus contain more RA-producing cells.
Addition of different concentrations of at-RA results in a dose-dependent linear response of the reporter cells. This response can be measured colorimetrically by reading the absorbance at 610 nm. The generated standard curve can then be used to quantify RA levels in samples, such as in wild-type neurosphere cultures (Figure 3A) or E8.5 embryos (Figure 3B).
<img alt="Figure 1" src="/files/ftp_upload/54443/54443fig1.jpg" /> 
Figure 1. Maintenance and Subcloning of F9 RARE-LacZ Cells. (A) A phase-contrast image of F9 RARE-LacZ cells is shown. These cells have a doubling time of ~10 hr and must be passed every 3 days (roughly 70 - 80% confluence) at a 1:10 ratio. (B) Periodic testing of cultures with addition of 1 nM at-RA and subsequent LacZ staining must be done to ensure cultures remain strong and uniform responders to RA (right). In case of poor and non-uniform responders (left), subcloning must be performed. Scale bars 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54443/54443fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54443/54443fig2.jpg" /> 
Figure 2. LacZ Staining of Co-cultured Samples (Dissociated E8.5 Embryos/Neurospheres) and F9 RARE-LacZ cells. (A) E8.5 mouse embryos of various genotypes were dissociated and plated on top of F9 RARE-LacZ cells. LacZ staining 24 hr later allows visualization of response of the F9 RARE-LacZ cells to endogenous RA produced by co-cultured samples. The Gpr161vl mutation results in decreased endogenous RA as visualized by the reduced response of the reporter cells. (B) Left. A phase-contrast image of neurospheres cultured from adult spinal cord neural stem cells is shown. Right. Neurospheres&#160;derived from adult Gpr161wt/wt spinal cord were dissociated and plated on top of F9 RARE-LacZ cells. The presence of blue precipitate following LacZ staining indicate the presence of endogenous RA in these neurospheres. The difference in staining intensity between neurosphere and E8.5 embryo co-cultures indicate greater levels of RA present in neurospheres than E8.5 embryos. Scale bars 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54443/54443fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54443/54443fig3.jpg" /> 
Figure 3. Standard Curve and Colorimetric Quantification of RA Levels. The response of the F9 RARE-LacZ reporter cell line can be measured colorimetrically at 610 nm using a standard ELISA plate reader. (A) Representative standard curve used to quantify relative RA levels of wild-type neurosphere cultures. (B) Quantification of relative RA levels from co-cultured E8.5 embryos. N= 3; *p&#60; 0.05, student&#39;s t-test. Error bars SEM. <a href="https://www.jove.com/files/ftp_upload/54443/54443fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stock Buffers/Solutions</td>
			<td>Components</td>
			<td>Storage conditions</td>
		</tr>
		<tr>
			<td>10% Sodium deoxycholate monohydrate (C24H39NaO4 &#183; H2O)</td>
			<td>10 ml in 100 ml dH2O</td>
			<td>4 &#186;C</td>
		</tr>
		<tr>
			<td>1 M magnesium chloride (MgCl2)</td>
			<td></td>
			<td>RT</td>
		</tr>
		<tr>
			<td>100x potassium ferrocyanide (K4[Fe(CN)6]&#183;3H6O)</td>
			<td>2.12 g in 10 ml dH2O</td>
			<td>RT in the dark</td>
		</tr>
		<tr>
			<td>100x potassium ferricyanide (K3[Fe(CN)6])</td>
			<td>1.64 g in 10 ml dH2O</td>
			<td>RT in the dark</td>
		</tr>
		<tr>
			<td>20 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-&#946;-d-galactopyranoside)</td>
			<td>add 5 ml dimethyl formamide to make 20 mg/ml stock solution</td>
			<td>-20 &#186;C</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>
		<tr>
			<td>Papain</td>
			<td>dissolve one vial in 7 ml HBSS</td>
			<td>4 &#186;C</td>
		</tr>
	</tbody>
</table>
Table 1. Buffers and Solution Compositions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.&#160;</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 G418 (final conc 0.4 mg/ml)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>to 500 ml DMEM/F-12 (with L-glutamine and 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>Neurosphere dissociation medium</td>
			<td>1 ml 20U papain</td>
			<td>prepare fresh every time</td>
		</tr>
		<tr>
			<td></td>
			<td>100 &#956;l 13 mg/ml trypsin</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 &#956;l 7 mg/ml hyaluronic acid</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>28 &#956;l 10 mg/ml DNaseI</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>50 &#956;l 4mg/ml kynurenic acid</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurosphere culture medium</td>
			<td>DMEM/F-12 (with L-glutamine and HEPES)</td>
			<td>prepare fresh every time</td>
		</tr>
		<tr>
			<td></td>
			<td>2% B-27 supplement</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>20 ng/ml fibroblast growth factor (FGF)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>20ng epidermal growth factor (EGF)</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% glutaraldehyde</td>
			<td>250 &#956;l 50% glutaraldehyde</td>
			<td>prepare fresh every time</td>
		</tr>
		<tr>
			<td></td>
			<td>4750 &#956;l 1x PBS</td>
			<td></td>
		</tr>
		<tr>
			<td>LacZ wash solution</td>
			<td>0.5 ml 10% deoxycholate (final conc 0.1%)</td>
			<td>Store at 4 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td>1.0 ml 100% NP-40 (final conc 0.2%)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>50 ml 10X PBS</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>distilled water to 500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal staining solution</td>
			<td>1x potassium ferrocyanide</td>
			<td>prepare fresh every time</td>
		</tr>
		<tr>
			<td></td>
			<td>1x potassium ferricyanide</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1 mg/ml X-gal</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>to appropriate volume using LacZ wash solution</td>
			<td></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 2. Working Buffers and Solution Compositions.

Watch this Scientific Journal Video about Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay at JoVE.co

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

quantitative-measurement-relative-retinoic-acid-levels-e85-embryos

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.8K Views.  Rutgers University. Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line. 

Video: Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

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 ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor &#947;-secretase-mediated cleavage of either APP-C99 or Notch, using a Gal4 promoter-driven firefly luciferase reporter system. These assays were established by stably co-transfecting HEK293 cells with the Gal4-driven luciferase reporter gene and either the Gal4/VP16-tagged C-terminal fragment of APP (APP-C99; CG cells), or the Gal4/VP16-tagged Notch-&#916;E (N&#916;E; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress &#947;-secretase cleavage of APP-C99 in CG cells, but not N&#916;E in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for &#947;-secretase modulators, and these responses are in stark contrast to the pan-inhibition of &#947;-secretase induced by DAPT. Our studies provide direct evidence that &#947;-secretase activities toward different substrates can be differentiated in a cellular context. These new assays may therefore be useful tools in drug discovery for improved AD therapies.

We have successfully generated two substrate-specific &#947;-secretase assays. Both cell-based assays presented here are designed to quantify &#947;-secretase enzymatic activities via the output of firefly luciferase reporters.

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...