measuring retinoic acid levels in neurospheres using a retinoic acid reporter cell line

Measuring Retinoic Acid Levels in Neurospheres Using a Retinoic Acid Reporter Cell Line

For neurosphere co-cultures, begin with transferring a neurosphere culture to 15-milliliter tubes. Spin the tubes at 200 g&#39;s for 10 minutes and remove all but 1 milliliter of the supernatant. Then triturate the contents with a P 1000 pipette.
Now, use a small volume to determine the density of the dissociated cells. Then, plate 100,000 dissociated neurosphere cells over the F9 RARE-LacZ cells. Load three wells with each neurosphere culture type. Now, be sure to prepare the wells for the standard curve.
In triplicate, add 100 microliters of all-trans RA solutions prepared by serial dilution at seven different concentrations in F9 RARE-LacZ culture medium. Include negative controls of untreated cells. Finally, culture the fully loaded 96-well plate overnight.
Remove the medium and fix the cultures using 100 microliters of 2.5% glutaraldehyde per well. Let the plate incubate at room temperature for 15 minutes. Later, remove the fixative and wash the wells twice with 200 microliters of PBS for 10 minutes per wash.
Next, wash the wells three times with 200 microliters of LacZ wash solution for 10 minutes per wash. During the third wash, prepare the X-gal staining solution in a 15-milliliter tube wrapped in foil. Also, prepare a humidified chamber for the 96-well plate.
Now, add 200 microliters of the X-gal staining solution to each well. Place the plate inside the humidified chamber, and incubate at 37 degrees Celsius. After the incubation, remove the LacZ staining solution and replace it with 200 microliters of PBS. Then, measure the absorbance at 610 nanometers and image the plate.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Solution and Media Preparation
<ol>
	<li>Prepare stock and buffer solutions as per&#160;Table 1. Prepare working solutions as per&#160;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&#160;Table 2.</li>
		<li>Coat 10 cm culture dishes (cell culture treated) with 10 ml&#160;of 0.2% gelatin (refer to&#160;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&#160;in a 37 &#176;C water bath for 2 - 3 min. Plate cells (around 1 x 106&#160;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 the supernatant and resuspend the cell pellet in 10 ml of F9 RARE-LacZ freezing medium (see&#160;Table 2). Aliquot 1 ml into labeled cryovials and transfer vials into freezing containers. Place the freezing container in -80 &#176;C freezer O/N and transfer the vials to liquid nitrogen tanks the next day.</li>
	</ol>
	</li>
</ol>
3. 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 faces 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 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 them 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&#160;Table 2). Put tube on ice and repeat steps 3.2.1 - 3.2.2 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 period 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&#160;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&#160;for 7 days until neurospheres appear.</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&#160;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>
4. Retinoic acid (RA) Assay of Spinal Cord Neurospheres
<ol>
	<li>Plating F9 RARE-LacZ in 96-well plate
	<ol>
		<li>One day prior to 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&#160;cells/well of F9-RARE LacZ cells, but this time using culture medium without G418.</li>
		<li>Prepare enough wells for the co-culture of neurospheres (3 wells/genotype) and for a standard curve in triplicate (27 wells).</li>
	</ol>
	</li>
	<li>Co-culture of F9 RARE-LacZ and Spinal Cord Neurospheres
	<ol>
		<li>Dissociate neurospheres and plate on top of F9 RARE-LacZ in the 96-well plate as detailed in step 3.4. 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&#160;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 4.1. Assign wells containing untreated F9 RARE-LacZ cells as the 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&#160;Table 2) for 15 min, RT. Remove the fixative and wash twice with 200 &#181;l 1x PBS, 10 min per wash.</li>
		<li>Remove PBS and wash three times with 200 &#181;l of the LacZ wash solution (see&#160;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&#160;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 for 4 - 8 hr to allow for development of blue-colored precipitate.</li>
	</ol>
	</li>
	<li>Reading Absorbance at OD610&#160;and Imaging
	<ol>
		<li>After the color reaction, remove the LacZ staining solution and replace it 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>
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>Stock Buffers/Solutions</td>
			<td>Components</td>
			<td>Storage conditions</td>
		</tr>
		<tr>
			<td>10% Sodium deoxycholate monohydrate (C24H39NaO4&#160;&#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 2. Working 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.</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 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>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 (K4[Fe(CN)6] &#183; 3H2O)</td>
			<td>Sigma Aldrich</td>
			<td>P-9387</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>potassium ferricyanide (K3[Fe(CN)6])</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>

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>&#160;J. Vis. Exp.&#160; (2016).
This video demonstrates the measurement of retinoic acid (RA) secretion from neurosphere cells using RA reporter cells. It outlines the steps involved in co-culturing neurosphere cells with RA reporter cells and performing a colorimetric RA assay to measure RA levels.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Solution and Media ...

Take an adherent culture of retinoic acid (RA) reporter cells.
These cells carry a transgenic construct containing the lacZ gene, which encodes &#946;-galactosidase, regulated by the retinoic acid response element (RARE).
Seed dissociated cells derived from mouse spinal cord stem cell neurospheres. Incubate.
The neurosphere cells release RA, which enters the reporter cells and binds to its receptor complex on RARE.
This triggers a transcriptional activator complex formation, initiating &#946;-galactosidase expression.
Replace the media with a fixative to preserve cellular morphology.
Remove the fixative and wash with a buffer.
Add a permeabilization buffer to permeabilize cellular membranes.
Introduce a &#946;-galactosidase substrate and incubate.
The substrate enters the reporter cells, where &#946;-galactosidase hydrolyzes it into an unstable product that oxidizes and dimerizes, forming a blue-colored precipitate.
Wash with a buffer.
Using a microplate reader, measure the absorbance and compare it to a standard curve to quantify RA secretion from neurosphere cells.

21 Views. Misurazione dei livelli di acido retinoico nelle neurosfere utilizzando una linea cellulare reporter di acido retinoico

Video: Misurazione dei livelli di acido retinoico nelle neurosfere utilizzando una linea cellulare reporter di acido retinoico - Experiment

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.

Controllo ottico di una proteina neuronale Utilizzando un acido Innaturale Amino geneticamente codificata in neuroni

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

Ricerca

JoVE Journal

Neuroscienze

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

Vivere-cella misura di Odorant recettore attivazione mediante un'analisi di campo in tempo reale

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

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.

Misurazione quantitativa della proteina precursore dell'amiloide γ-secretasi-mediata e la fenditura di Notch in Cell-based Luciferase Reporter Assay piattaforme

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

Measuring Retinoic Acid Levels in Neurospheres Using a Retinoic Acid Reporter Cell Line

Trascrizione

Measuring Retinoic Acid Levels in Neurospheres Using a Retinoic Acid Reporter Cell Line