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

Abstract

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 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.

Protocol

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

1. Solution and Media Preparation

1. Prepare stock and buffer solutions as per Table 1. Prepare working solutions as per Table 2.

2. Culture and Maintenance of F9 RARE-LacZ Cells

1. Thawing Frozen Cells
   1. Prepare F9 RARE-LacZ cell culture medium as detailed in Table 2.
   2. 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.
   3. Thaw a vial of F9 RARE-LacZ cells in a 37 °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 °C, 5% CO₂. Change the medium the next day.
2. Maintenance of F9 RARE-LacZ Cells
   1. 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.
   2. 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.
3. Freezing F9 RARE-LacZ Cells
   1. 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.
   2. Remove the supernatant and resuspend the 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 the freezing container in -80 °C freezer O/N and transfer the vials to liquid nitrogen tanks the next day.

3. Laminectomy, Dissection of Adult Lumbar Spinal Cord and Neurosphere Culture

1. Laminectomy
   1. Sacrifice an adult (P60) mouse through cervical dislocation.
   2. 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.
   3. 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.
   4. 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'clock and 9 o'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.
   5. 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.
2. Spinal Cord Dissection
   1. Pour the culture medium with the spinal cord into a 6 cm dish (non-cell culture treated).
   2. 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.
   3. Transfer minced tissue to 15 ml tube containing 1 ml of neurosphere dissociation media (see Table 2). Put tube on ice and repeat steps 3.2.1 - 3.2.2 until all spinal cords have been processed.
   4. Incubate tubes containing minced tissue in dissociation medium at 37 °C for 10 min, flick the tube on the side to mix, and then incubate at 37 °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: "regular", "fine", and "extra fine". For "regular" 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 "fine" (~ >1 mm and > 5 mm diameter) and "extra fine" (~ 0.5 mm) polished pipettes. Do not use pipettes with diameters less than 0.5 mm.
3. Neurosphere Culture
   1. 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.
   2. Transfer 10 µl 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 °C and 5% CO₂ for 7 days until neurospheres appear.
4. Neurosphere Dissociation
   1. 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 µl of dissociated cells into a hemocytometer and get a cell count.
   2. 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 °C and 5% CO₂.

4. Retinoic acid (RA) Assay of Spinal Cord Neurospheres

1. Plating F9 RARE-LacZ in 96-well plate
   1. One day prior to dissociation of neurospheres, coat a 96-well plate with 100 µl of 0.2% gelatin per well for 30 min at RT. Aspirate out the gelatin and rinse twice with 200 µl of distilled water.
   2. 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.
   3. Prepare enough wells for the co-culture of neurospheres (3 wells/genotype) and for a standard curve in triplicate (27 wells).
2. Co-culture of F9 RARE-LacZ and Spinal Cord Neurospheres
   1. 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 °C and 5% CO₂.
3. Generating a Standard Curve
   1. 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.
   2. Add 100 µl 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 °C and 5% CO₂.
      Note: F9 RARE-LacZ cells respond in a dose-dependent linear manner to varying concentrations of all-trans RA.
4. RA Assay
   1. 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 µl of 2.5% glutaraldehyde (see Table 2) for 15 min, RT. Remove the fixative and wash twice with 200 µl 1x PBS, 10 min per wash.
   2. Remove PBS and wash three times with 200 µl 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 µl/well).
      Note: X-gal staining solution is light-sensitive. Use the staining solution within 15 min after preparation.
   3. 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 µl of the X-gal staining solution per well of the 96-well plate and place the plate in a humidified chamber.
   4. Incubate humidified chamber containing the 96-well plate at 37 °C for 4 - 8 hr to allow for development of blue-colored precipitate.
5. Reading Absorbance at OD₆₁₀ and Imaging
   1. After the color reaction, remove the LacZ staining solution and replace it with 200 µl 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.

Table 1. Buffers and Solution Compositions.

Stock Buffers/Solutions	Components	Storage conditions
10% Sodium deoxycholate monohydrate (C24H39NaO4 · H2O)	10 ml in 100 ml dH2O	4 ºC
1 M magnesium chloride (MgCl2)		RT
100x potassium ferrocyanide (K4[Fe(CN)6]·3H6O)	2.12 g in 10 ml dH2O	RT in the dark
100x potassium ferricyanide (K3[Fe(CN)6])	1.64 g in 10 ml dH2O	RT in the dark
20 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside)	add 5 ml dimethyl formamide to make 20 mg/ml stock solution	-20 ºC
10 mM (3 mg/ml) all-trans retinoic acid	Dissolve 50 mg in 16.67 ml absolute ethanol. Prepare 1 ml aliquots.	-80 ºC in the dark, in 1.5 ml amber microcentrifuge tubes
Papain	dissolve one vial in 7 ml HBSS	4 ºC

Table 2. Working Buffers and Solution Compositions.

Working Buffers/Solutions	Components	Storage conditions
F9 RARE-LacZ cell culture medium	50 ml fetal bovine serum (FBS) (final concentration 10%)	Filter-sterilize.
	5 ml 100x Penicillin Streptomycin (final conc 1X)	Store at 4 ºC.
	4 ml 50 mg/ml G418 (final conc 0.4 mg/ml)
	to 500 ml DMEM/F-12 (with L-glutamine and HEPES)
0.2% gelatin	25 ml 2.0% gelatin	Store at 4 ºC.
	80 ml distilled water
	Mix well
70% ethanol	30 ml 95% ethanol	Place in spray bottle.
	70 ml distilled water	Store at RT
Neurosphere dissociation medium	1 ml 20U papain	prepare fresh every time
	100 μl 13 mg/ml trypsin
	100 μl 7 mg/ml hyaluronic acid
	28 μl 10 mg/ml DNaseI
	50 μl 4mg/ml kynurenic acid
Neurosphere culture medium	DMEM/F-12 (with L-glutamine and HEPES)	prepare fresh every time
	2% B-27 supplement
	20 ng/ml fibroblast growth factor (FGF)
	20ng epidermal growth factor (EGF)
2.5% glutaraldehyde	250 μl 50% glutaraldehyde	prepare fresh every time
	4750 μl 1x PBS
LacZ wash solution	0.5 ml 10% deoxycholate (final conc 0.1%)	Store at 4 ºC.
	1.0 ml 100% NP-40 (final conc 0.2%)
	50 ml 10X PBS
	distilled water to 500 ml
X-gal staining solution	1x potassium ferrocyanide	prepare fresh every time
	1x potassium ferricyanide
	1 mg/ml X-gal
	to appropriate volume using LacZ wash solution
F9 RARE-LacZ freezing medium	F9 RARE-LacZ culture medium + 5% DMSO	prepare fresh every time

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C3H/HeSn-Gpr161vl/J mouse strain	Jackson Laboratory	316
F9 RARE-LacZ reporter cell line			from Dr. Michael Wagner (SUNY Downstate Medical Center) and Ben Thiede (Dr. Jeff Corwin lab, University of Virginia)
DMEM/F-12 (with L-glutamine and HEPES)	ThermoFisher Scientific	11330-057
Fetal Bovine Serum (FBS), qualified	ThermoFisher Scientific	26140-079	Store at -20 °C in 10-ml aliquots to avoid repeated freeze-thaw; warm in 37 C for media preparation
Penicillin/Streptomycin	ThermoFisher Scientific	15140-122	Store at -20 °C in 10-ml aliquots to avoid repeated freeze-thaw; light-sensitive
G418	ThermoFisher Scientific	10131-027	Store at 4 °C; light-sensitive
2% Gelatin	Sigma Aldrich	G1393	Store at 4 °C
B-27 supplement (2 % stock solution)	ThermoFisher Scientific	17504-044	Store at -20 °C in 1-ml aliquots to avoid repeated freeze-thaw; warm at room temperature for media preparation
Murine Epidermal Growth Factor (EGF)	PeproTech	315-09	Prepare 100 μg/ml stock solution in 0.1% bovine serum albumin (BSA) in 1X PBS; prepare working concentrations by diluting with 1X PBS; store at -20 °C
Murine Fibroblast Growth Factor (FGF)-basic	PeproTech	450-33	Prepare 100 μg/ml stock solution in 0.1% bovine serum albumin (BSA) in 1X PBS; prepare working concentrations by diluting with 1X PBS; store at -20 °C
50% glutaraldehyde	Amresco	M155	Store at 4 C
Sodium deoxycholate monohydrate (C24H39NaO4 · H2O)	Sigma Aldrich	D5670	Store at room temperature
potassium ferrocyanide (K4[Fe(CN)6] · 3H2O)	Sigma Aldrich	P-9387	Store at room temperature
potassium ferricyanide (K3[Fe(CN)6])	Sigma Aldrich	P-8131	Store at room temperature
X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside)	Promega	V3941	Store stock at -20 °C
Dimethyl sulfoxide (DMSO)	Sigma Aldrich	41639	Store at room temperature
all-trans Retinoic Acid (at-RA)	Sigma Aldrich	R-2625	Store 1 ml aliquots of stock in -80 °C; extremely sensitive to light
TrypLE Express	ThermoFisher Scientific	12605-010	Store at room temperature
0.25% trypsin-EDTA	ThermoFisher Scientific	25200-056	alternative to TrypLE Express; aliquot and store at -20 C
Hank's Balanced Salt Solution (HBSS)	ThermoFisher Scientific	14170-112	Store at 4 C
10X phosphate buffered solution (PBS)	ThermoFisher Scientific	10010-023	Store at 4 C
Papain	Worthington	LK003176	Store at 4 °C.
Trypsin	Worthington	LS003703	Store in 1 ml aliquots at -20 °C.
Hyaluronic acid	Calbiochem	385908	Store 200 μl aliquots at -20 °C.
DNaseI	Worthington	LS002139	Store in 200 μl aliquots at -20 C
Kynurenic acid	Sigma Aldrich	K3375	Store in 200 μl aliquots at -20 C
95% ethanol
Mr. Frosty Freezing container	ThermoFisher Scientific	5100-0001
10 cm culture dish (non-cell culture treated)
10 cm culture dish (cell culture treated)
6 cm culture dish (non-cell culture treated)
96-well cell culture plates
1.5 ml microcentrifuge tubes
1.5 ml amber microcentrifuge tubes
1.5 ml cryovials
37 °C water bath
surgical scissors
fine surgical scissors
no. 5 forceps
blunt-ended forceps
scalpel blade
glass pasteur pipettes
cotton
alcohol lamp
ELISA plate reader
stereomicroscope