Luciferase Assay for Measuring γ-Secretase Activity in Human Cells

Begin with genetically modified human cells in a growth medium.

These cells contain a tetracycline-inducible system possessing the amyloid precursor protein or APP gene and a luciferase gene.

Exchange with a medium containing tetracycline and incubate.

Tetracycline binds to the activator protein, which binds to the APP promoter.

This induces gene expression and produces the precursor proteins that localize to the cell membrane.

The β-Secretase enzyme cleaves APP, generating a fragment that remains attached to the cell membrane.

The γ-secretase cleaves this fragment, producing amyloid-β peptides and releasing an activator component.

This activator enters the nucleus, binds to the luciferase promoter, induces gene expression, and produces luciferase enzymes.

Replace the medium with an assay reagent containing the luciferase substrate. The substrate diffuses into the cells and reacts with luciferase, producing bioluminescence.

Use a microplate reader to measure the light intensity, which directly reflects γ-secretase activity, an enzyme crucial in neuropathological conditions.

Count NG or CG cells with a hemocytometer. Then, seed the cells at 20,000 cells per well onto 96-well micropl