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

Take an adherent culture of retinoic acid (RA) reporter cells.

These cells carry a transgenic construct containing the lacZ gene, which encodes β-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 β-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 β-galactosidase substrate and incubate.

The substrate enters the reporter cells, where β-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.

For neurosphere co-cultures, begin with transferring a neurosphere culture to 15-milliliter tubes. Spin the tubes at 200 g'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.