Luciferase Assay for Measuring γ-Secretase Activity in Human Cells

Samenvatting

Source: Wang, B., et al., Quantitative Measurement of γ-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms. J. Vis. Exp. (2018).

This video demonstrates the procedure of measuring γ-secretase activity in genetically modified human cells using a tetracycline-inducible system. The process involves inducing APP gene expression with tetracycline, generating amyloid-β peptides, and activating luciferase production. The bioluminescence resulting from the luciferase-substrate reaction is then measured, reflecting γ-secretase activity.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 

1. Measurement of Luciferase Reporter Signals, which Correspond to γ-Secretase Cleavage of APP-C99 (amyloid precursor protein - C-terminal 99-amino acid fragment)

NOTE: Please refer to the previous publications for detailed descriptions of the generation of CG (C99-Gal4) cells.

1. Seed CG cells (20, 000 cells/well) onto 96-well microplates at a final volume of 200 μL/well in growth medium that is composed of Dulbecco's Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS), 5 μg/mL blasticidin, 250 μg/mL antibiotic (e.g., zeocin), and 200 μg/mL hygromycin B.
   1. Count cells with a hemocytometer.
   2. Transfer the microplates to a humidified CO₂ incubator and incubate at 37 °C overnight.
2. Remove 100 μL of growth medium from each well.
3. Add 100 μL/well of growth medium containing 2 μg/mL tetracycline with either 0.2% dimethyl sulfoxide (DMSO), 2 μM CL-387,785, 2 μM N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT), or other related ErbB1/ErbB2 inhibitors.
4. Incubate treated CG cells in microplates at 37 °C for 24 h.
5. Remove 150 μL/well growth medium from microplates.
6. Add 50 μL/well luciferase assay reagent (please see Table of Materials).
7. Transfer the microplates to a luminescence microplate reader.
   1. Maintain the microplates at room temperature for 5 min with gentle agitation.
8. Determine the Firefly luciferase (FL)-emitted luminescence using a pre-defined program stored in the luminescence microplate reader.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
VictorLight luminescence plate reader	PerkinElmer	2030-0010
Class II, Type 2 Biological Safety Cabinet	Thermo Scientific	1300 Series A2
CL-387,785	EMD Calbiochem	233100-1MG
DAPT	Merck	565770
Dulbecco's Modified Eagle Medium (DMEM)	Thermo Fisher	12100046	main compnent of growth medium
Fetal bovine serum (FBS)	Thermo Fisher	16000044
Blasticidin	Thermo Fisher	A1113903
Zeocin	Thermo Fisher	R25005
Hygromycin B	Gibco	10687010
Hemocytomer	Sigma-Aldrich	BR717805 Aldrich
96-well microplate	Nunc	156545
Tetracycline	Sigma	T7660
Dimethyl sulfoxide (DMSO)	Sigma	D2650
Steady-Glo luciferase assay reagent	Promega	E2510
Humidified CO2 incubator	Revco	Ultima II
T-REx293 cell line	Invitrogen	R71007
Wallac 1420 software version 3.0	PerkinElmer		instrument control software of the luminescence microplate reader