This article describes a method for measuring ��-secretase activity using genetically modified human cells. The process involves inducing gene expression of the amyloid precursor protein (APP) and luciferase, followed by quantifying bioluminescence as a measure of enzyme activity.
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 microplates at a final volume of 200 microliters per well in growth medium. Transfer the microplates to a humidified carbon dioxide incubator, and incubate at 37 degrees Celsius overnight. Following overnight incubation, remove 100 microliters of growth medium from each well.
Add 100 microliters per well of growth medium containing 2 micrograms per milliliter of tetracycline with either 0.2% dimethylsulfoxide, two micromolar CL 387, 785, two micromolar DAPT or other related ErbB1/ErbB2 inhibitors. Incubate the treated cells in microplates at 37 degrees Celsius for 24 hours. Following incubation, remove 150 microliters per well of growth medium from the microplates.
Then add 50 microliters per well of luciferase assay reagent. Next, transfer the microplates to a luminescence microplate reader and maintain them at room temperature for five minutes. Determine the firefly luciferase emitted luminescence using a predefined program stored in the luminescence microplate reader.
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