luciferase assay for measuring &#947-secretase activity in human cells

Luciferase Assay for Measuring &#947-Secretase Activity in Human Cells

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.

luciferase-assay-for-measuring-947-secretase-activity-in-human-cells

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

Luciferase Assay for Measuring &#947;-Secretase Activity in Human Cells

EoE Sub Articles

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.&#160;
1. Measurement of Luciferase Reporter Signals, which Correspond to &#947;-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.
<ol>
	<li>Seed CG cells (20, 000 cells/well) onto 96-well microplates at a final volume of 200 &#956;L/well in growth medium that is composed of Dulbecco&#39;s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS), 5 &#956;g/mL blasticidin, 250 &#956;g/mL antibiotic (e.g., zeocin), and 200 &#956;g/mL hygromycin B.
	<ol>
		<li>Count cells with a hemocytometer.</li>
		<li>Transfer the microplates to a humidified CO2 incubator and incubate at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Remove 100 &#956;L of growth medium from each well.</li>
	<li>Add 100 &#956;L/well of growth medium containing 2 &#956;g/mL tetracycline with either 0.2% dimethyl sulfoxide (DMSO), 2 &#956;M CL-387,785, 2 &#956;M N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT), or other related ErbB1/ErbB2 inhibitors.</li>
	<li>Incubate treated CG cells in microplates at 37 &#176;C for 24 h.</li>
	<li>Remove 150 &#956;L/well growth medium from microplates.</li>
	<li>Add 50 &#956;L/well luciferase assay reagent (please see Table of Materials).</li>
	<li>Transfer the microplates to a luminescence microplate reader.
	<ol>
		<li>Maintain the microplates at room temperature for 5 min with gentle agitation.</li>
	</ol>
	</li>
	<li>Determine the Firefly luciferase (FL)-emitted luminescence using a pre-defined program stored in the luminescence microplate reader.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>VictorLight luminescence plate reader</td>
			<td>PerkinElmer</td>
			<td>2030-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II, Type 2 Biological Safety Cabinet</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td></td>
		</tr>
		<tr>
			<td>CL-387,785</td>
			<td>EMD Calbiochem</td>
			<td>233100-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Merck</td>
			<td>565770</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher</td>
			<td>12100046</td>
			<td>main compnent of growth medium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Thermo Fisher</td>
			<td>A1113903</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Thermo Fisher</td>
			<td>R25005</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Gibco</td>
			<td>10687010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytomer</td>
			<td>Sigma-Aldrich</td>
			<td>BR717805 Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>Nunc</td>
			<td>156545</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Sigma</td>
			<td>T7660</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Steady-Glo luciferase assay reagent</td>
			<td>Promega</td>
			<td>E2510</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified CO2 incubator</td>
			<td>Revco</td>
			<td>Ultima II</td>
			<td></td>
		</tr>
		<tr>
			<td>T-REx293 cell line</td>
			<td>Invitrogen</td>
			<td>R71007</td>
			<td></td>
		</tr>
		<tr>
			<td>Wallac 1420 software version 3.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>instrument control software of the luminescence microplate reader</td>
		</tr>
	</tbody>
</table>

luciferase assay for measuring &#947;-secretase activity in human cells

Source: Wang, B., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56795">Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms.</a> J. Vis. Exp. (2018).
This video demonstrates the procedure of measuring &#947;-secretase activity in genetically modified human cells using a tetracycline-inducible system. The process involves inducing APP gene expression with tetracycline, generating amyloid-&#946; peptides, and activating luciferase production. The bioluminescence resulting from the luciferase-substrate reaction is then measured, reflecting &#947;-secretase activity.

Source: Wang, B., et al., Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase ...

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 &#946;-Secretase enzyme cleaves APP, generating a fragment that remains attached to the cell membrane.
The &#947;-secretase cleaves this fragment, producing amyloid-&#946; 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 &#947;-secretase activity, an enzyme crucial in neuropathological conditions.

luciferase-assay-for-measuring-secretase-activity-in-human-cells

16 visualizações. Luciferase Assay for Measuring &#947-Secretase Activity in Human Cells

Vídeo: Luciferase Assay for Measuring &#947-Secretase Activity in Human Cells - Experimento

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Imagem de tecidos amiloides manchado com Luminescent Oligothiophenes conjugados por hiperespectrais Confocal imagem de vida de fluorescência e microscopia

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

JoVE Journal

Bioquímica

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

Visualização dos depósitos β amiloide no cérebro humano com Matrix-assisted Laser dessorção/ionização de imagem espectrometria de massa

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Neurociência

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.
While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the &#947;-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.
We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

Imaging o tráfego intracelular de APP com GFP fotoactivável

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

Luciferase Assay for Measuring γ-Secretase Activity in Human Cells

Transcrição

Luciferase Assay for Measuring γ-Secretase Activity in Human Cells