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

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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.

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

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