The overall goal of this quantitative cell-based reporter gene assay for gamma-secretase is to examine the differential effect of putative gamma-secretase modulators on gamma-secretase-mediated cleavage. This method can help to answer the key questions in the Alzheimer disease field, such as identification of novel gamma-secretase modulators for the treatment of Alzheimer disease. The advantage of this technique is that it is a highly efficient assay system, and it can easily identify gamma-secretase modulators.
The implication of this technique extend toward therapy of Alzheimer disease through the potential discovery of anti-AD drugs with minimal side effects. This method can provide insight to the substrate selectivity of gamma-secretase. It also can be applied to the studies of other diseases, such as neuroblastoma.
Individual new to this method may struggle with careful removal and addition of cell culture media to 96 microplate, as too much disturbance may complicate the final results. Begin this procedure with preparation of NG and CG cells, as referenced in the text protocol. 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 two micrograms per milliliter of tetracycline with either 0.2%dimethyl sulfoxide, two micromolar CL-387, 785, two micromolar DAPT, or other related ErbB1/ErbB2 inhibitors. Incubate the treated cells and 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.
First, open the instrument control software. Then, from the Tool menu, select Luminometry. Under the Parameters setting, choose No Filter for emission filter, tick Normal for emission aperture, enter one for counting time, and click on OK to set up the luminescence reading.
Under the Instrument control tab, scroll through the list of active protocols, and select the pre-stored protocol for reading luminescence. Under the Live display tab, define the scale from the pull-down menu, and select Logarithm or Linear for the type. Under the Temperature tab, select Off for plate heating.
Finally, click the Start button to perform the luminescence reading. Export the results into a spreadsheet format using the Explorer embedded within the control software. Estimate background luminescence from CG or NG cells treated with growth medium that does not include tetracycline.
Define a luminescence signal emitted by CG or NG cells treated with 0.1%DMSO in growth medium containing one microgram per milliliter tetracycline alone as 100%relative gamma-secretase activity. Finally, normalize luciferase signals from CG or NG cells that are cultured with growth medium containing tetracycline and either CL-387, 785 or DAPT to the luciferase signal emitted by DMSO-treated CG or NG cells. Shown here, are representative results demonstrating the quantitative measurements of gamma-secretase cleavage of APP-C99 in the CG cells.
Cells were treated with one micromolar of CL-387, 785 or DAPT in the presence of one microgram per milliliter tetracycline at 37 degrees Celsius for 24 hours. The luminescence derived from gamma-secretase-mediated proteolysis of C99-GV was quantified after the addition of 100 microliters per well of luciferase assay reagent. The luciferase signal emitted by cells treated with vehicle alone was set as 100%relative luminescence.
Representative results demonstrating the quantitative measurements of gamma-secretase cleavage of N-delta-E in the NG cells are shown here. Similarly, cells were treated with one micromolar of CL-387, 785 or DAPT in the presence of one microgram per milliliter tetracycline at 37 degrees Celsius for 24 hours. The luminescence derived from gamma-secretase-mediated proteolysis of N-delta-E-GV was quantified after the addition of 100 microliters per well of luciferase assay reagent.
The luciferase signal emitted by cells treated with vehicle alone was set as 100%relative luminescence. Once mastered, this technique only requires a hands-on time of one to two hours, pending the number of compounds to exam. After its development, this technique paved the way for the researcher in the field of Alzheimer disease to explore genetic and chemical modifier of gamma-secretase in pursuit of novel therapeutics for Alzheimer disease.
