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NOTE: The following protocols describe the sample preparation and the quantification of neutrophil serine proteases (NSPs) activity. The experimental procedures presented herein focus on human sputum and neutrophil elastase (NE) or cathepsin G (CG) activity measurement. However, slight adaptations in the sample preparation protocol render the analysis of blood-derived cells and tumor homogenates possible. In addition, matrix metalloproteinase and cathepsin S activities can be investigated similarly by means of dedicated FRET probes
1. Sample preparation: cell isolation and supernatant separation
NOTE: If possible, the treatment of sputum should be carried out within 120 min after expectoration and the sputum should be stored on ice until further processing.
<ol>
	<li>If spontaneous expectoration of sputum is not possible, induce sputum as described previously. Briefly, inhale 200 &#181;g of the &#946;-2-receptor-antagonist salbutamol before starting the sputum induction procedure. Afterward, inhale a hypertonic (6%) saline solution for 15 minutes using a nebulizer. Collect the expectorated sputum in a Petri dish.</li>
	<li>Separate the mucus clumps from the saliva into a Petri dish with the help of a pipette tip.</li>
	<li>Weigh the mucus. 
	NOTE: The average weight of a mucus sample is 0.8 g (varying between 0.1 g and 5 g); 0.1 g is usually sufficient to perform the mentioned procedures.</li>
	<li>Add 4 parts (v/w) of 10% Sputolysin (in PBS) to sputum (for example, 4 mL of 10% Sputolysin for each gram of sputum). 
	CAUTION: Sputolysin is composed of concentrated dithiothreitol in phosphate buffer, therefore handle it with care.</li>
	<li>Incubate the mixture at room temperature (RT) on a rocking shaker for 15 min to dissolve the mucus. For safety reasons, place the shaker into a fume hood.</li>
	<li>Quench the reaction by adding the same volume of cold PBS (for example: 1 mL of cold PBS for each mL of 10% Sputolysin).</li>
	<li>Mix by pipetting to obtain a homogenous solution.</li>
	<li>Filter the mixture through a 100 &#181;m nylon cell strainer into a 50 mL tube.</li>
	<li>Repeat the filtration step through a 40 &#181;m nylon cell strainer.</li>
	<li>Centrifuge the solution for 10 min at 300 x g at 4 &#176;C.</li>
	<li>Transfer the supernatant fraction carefully into a fresh tube and store it on ice. 
	NOTE: The supernatant fractions can be stored at -20 &#176;C or -80 &#176;C until further analysis.</li>
	<li>Gently resuspend the cell pellet in 500 &#181;L of cold PBS and place it on ice. 
	&#8203;NOTE: The cell fraction must be processed immediately.</li>
</ol>
2. Neutrophil serine protease activity measurement
NOTE: Here, different methods are introduced to quantify NSP&#39;s activity by means of FRET reporters. The choice of the technology is dictated by the specific biomedical question and purpose of the experiment. The probes presented were extensively tested for their specificity against a set of lung-relevant enzymes. Although the probes are specific toward their target enzyme, always check the probe specificity on the clinical sample of interest. This can be achieved by incubating the sample with a specific protease inhibitor prior to probe addition, which should abolish any increase in the donor/acceptor (D/A) ratio.
<ol>
	<li>Soluble NSP&#39;s activity quantification via fluorimeter or plate reader assay 
	&#8203;NOTE: Protease activity in soluble fractions of the sample can be detected with any instrument capable of fluorescence detection.
	<ol>
		<li>Thaw enzymes on ice.</li>
		<li>Until use, keep NE and CG in an acidic storage buffer (50 mM sodium acetate, 200 mM NaCl, pH 5.5) to prevent self-cleavage.</li>
		<li>To set up an enzyme standard curve, prepare a 1:2 serial dilution of the enzyme (33.9 - 0.271 nM for NE; 42.6 - 0.333 nM for CG) in activation buffer (10 mM Tris-HCl, 500 mM NaCl at pH 7.5). The activation buffer has a neutral pH and therefore enables enzymatic catalysis to occur efficiently.
		<ol>
			<li>To prepare the highest standard concentration (NE concentration: 33.9 nM), dilute 1 &#181;L of NE (33.9 &#181;M) in 999 &#181;L of activation buffer.</li>
			<li>To prepare the second standard (NE concentration: 16.95 nM), mix 200 &#181;L of the first dilution with 200 &#181;L of activation buffer.</li>
			<li>Proceed accordingly to prepare the remaining 1:2 dilutions.</li>
			<li>The last standard, which is the blank, is composed of a pure activation buffer. The measurement of technical duplicates or triplicates is recommended. During the standard and sample preparation, try to keep vials on ice.</li>
		</ol>
		</li>
		<li>In parallel to the standard preparation, dilute sputum samples in an activation buffer. Dilute the human samples before assessing their protease activity to remain in the linear range of increase of reporter signal (D/A ratio). If patient samples were left undiluted, the cleavage would happen too rapidly for a reliable fitting. Since healthy donor sputum contains fewer active proteases compared to samples from cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients, different dilutions are generally performed (1:10 for healthy sputum supernatant, 1:20-500 for sputum supernatant of COPD or CF patients). 
		NOTE: In order to quantitatively measure protease activity in samples where their concentration is unknown, a standard curve with known enzyme concentrations needs to be measured in parallel, ideally on the same plate. The concentration of active enzyme in human sputum is calculated via interpolating the slopes measured in human sputum samples with the ones measured with the standard curves.</li>
		<li>Before preparing the samples for measurement, set up the instrument. Set the excitation wavelength for the NE F&#246;rster resonance energy transfer-based (FRET) probe (NEmo-1) to 354 nm, and set the detection wavelength to 400 nm for the donor and 490 nm for the acceptor. Set the excitation wavelength for CG FRET probe (sSAM) to 405 nm, and set the emission to 485 (donor) and 580 nm (acceptor).</li>
		<li>Add 40 &#181;L of samples, standard or blank into the wells of a black 96-well half area plate.</li>
		<li>To prepare the master mix containing the reporters (concentration of the reporter in the master mix: 10 &#181;M), dilute the probe stock (1 mM in dimethylsulfoxide, DMSO) 1:100 in activation buffer. Prepare the needed master mix volume by multiplying 10 &#181;L x the number of required plate wells. To reach the optimal final concentration (2 &#181;M) for fluorescence measurement of NEmo-1 and sSAM reporters, add 10 &#181;L of the master mix to each well (containing 40 &#181;L of either sample, standard or blank) and start the readout. NEmo-1 and sSAM reporters will therefore monitor soluble neutrophil elastase and cathepsin G activity, respectively. 
		NOTE: If a reagent injector is not available, make sure to start the readout as soon as possible after the reporter addition to the samples.</li>
		<li>Start the plate reader measurement and record the D/A ratio increase every 60-90 seconds for at least 20 min or until the increase in the signal reaches a plateau.</li>
		<li>Once data are exported, calculate the D/A ratio by dividing the donor relative fluorescence units (RFU) with the acceptor RFU for each time point and sample.</li>
		<li>Calculate the D/A ratio mean and standard deviation of each sample.</li>
		<li>Determine the slope within the linear growth of the D/A ratio change. The slope is an indicator of the enzyme cleavage rate for a FRET probe. Calculate the concentration of active enzyme in sputum by fitting the linear regression slopes derived from the human samples with the ones calculated from the enzyme standard.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 &#181;m Nylon cell strainer</td>
			<td>Corning Inc.</td>
			<td>431752</td>
			<td></td>
		</tr>
		<tr>
			<td>2300 EnSpire (Multilabel Plate Reader)</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35x10mm Dish, Nunclon Delta</td>
			<td>Thermo Fisher Scientific</td>
			<td>150318</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Nylon cell strainer</td>
			<td>Corning Inc.</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Sarstedt</td>
			<td>10535253</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>OHAUS Instruments (Shanghai) Co., Ltd.</td>
			<td>PR124</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon Round-Bottom Tubes 5 mL</td>
			<td>BD Bioscience</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRFortessa cell analyzer</td>
			<td>BD Bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>black flat bottom 96 well half area plate</td>
			<td>Corning Life Science</td>
			<td>3694</td>
			<td></td>
		</tr>
		<tr>
			<td>Cathepsin G</td>
			<td>Elastin Products Company</td>
			<td>SG623</td>
			<td></td>
		</tr>
		<tr>
			<td>Cathepsin G Inhibitor I</td>
			<td>Merck KGaA</td>
			<td>219372</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5418R</td>
			<td>Eppendorf AG</td>
			<td>EP5401000137</td>
			<td></td>
		</tr>
		<tr>
			<td>Combitips advanced 1.0 mL</td>
			<td>Eppendorf AG</td>
			<td>0030 089 430</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete proteinase inhibitor</td>
			<td>Roche</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Sputum Leucocyte Elastase</td>
			<td>Elastin Products Company</td>
			<td>SE563</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipette plus</td>
			<td>Eppendorf AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NEmo-1</td>
			<td>SiChem</td>
			<td>SC-0200</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>NEmo-2E</td>
			<td>SiChem</td>
			<td>SC-0201</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Pari Boy SX with an LC Sprint jet nebulizer</td>
			<td>Pari</td>
			<td>085G3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Gibco</td>
			<td>10010-015</td>
			<td></td>
		</tr>
		<tr>
			<td>ROTI Histokitt (mounting medium)</td>
			<td>Carl Roth GmbH + Co.KG</td>
			<td>6638.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Salbutamol</td>
			<td>Teva GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sivelestat</td>
			<td>Cayman Chemicals</td>
			<td>17779</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputolysin</td>
			<td>Calbiochem</td>
			<td>560000-1SET</td>
			<td></td>
		</tr>
		<tr>
			<td>sSAM</td>
			<td>in house</td>
			<td></td>
			<td>2 mM</td>
		</tr>
	</tbody>
</table>

an in vitro assay for measuring neutrophil serine protease activity using a fluorescent reporter

Source: Frey, D. L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62193">Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples.</a> J. Vis. Exp. (2021).
This video demonstrates an in vitro assay to quantify neutrophil serine protease activity in sputum samples. The sample containing secreted protease is incubated with a fluorescent reporter bearing a recognition motif. Cleavage of the motif by the protease enables individual fluorescence emission by the donor and acceptor fluorophore of the reporter, indicating protease activity in the sample.

An In Vitro Assay for Measuring Neutrophil Serine Protease Activity Using a Fluorescent Reporter

NOTE: The following protocols describe the sample preparation and the quantification of neutrophil serine proteases (NSPs) activity. The experimental ...

Take a microplate containing neutrophil serine proteases or NSPs. These soluble enzymes with proteolytic activity, secreted by neutrophils, function in inflammatory responses.
Add fluorescent reporter probes carrying a recognition motif that acts as a cleavage site for NSPs. The probes are conjugated to a donor and an acceptor fluorophore.
Upon excitation by a specific wavelength, the donor fluorophore emits energy. Due to its proximity to the donor, the acceptor fluorophore absorbs the energy and emits fluorescence of a different wavelength.
Detect the donor and acceptor signals separately and compute the donor-to-acceptor ratio to assess NSP activity. A low ratio indicates quenching of donor fluorescence in the absence of probe cleavage.
The NSPs recognize and cleave the recognition motif on the probes, releasing the acceptor.
The distance from the acceptor enables fluorescence emission from the donor fluorophore, increasing the donor-to-acceptor ratio. A high ratio indicates efficient probe cleavage by the NSPs.

an-vitro-assay-for-measuring-neutrophil-serine-protease-activity

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

62 ビュー. 出典:Frey, D. L., et al. ヒト喀痰サンプル中の好中球エラスターゼおよびカテプシンG活性のモニタリング. J. Vis. Exp.(2021年)。 このビデオは、喀痰サンプル中の好中球セリンプロテアーゼ活性を定量化するin vitroアッセイを示しています。分泌されたプロテアーゼを含むサンプルは、認識モチーフを持つ蛍光レポーターとインキュベートされます。プロテアーゼによるモチーフ?...

ビデオ: 蛍光レポーターを用いた好中球セリンプロテアーゼ活性測定のためのin vitroアッセイ - 概念

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

蛍光ペプチド ザイモグラフィーによってプロテアーゼ活性の検出

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

JoVE Journal

がん研究

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

シングル売り上げ高プロテアーゼ PCSK9 のタンパク質分解を評価する高スループット ルシフェラーゼ アッセイ

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

生化学

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

非侵襲的診断のプロテアーゼ活動In Vivo検出するナノセンサー

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

An In Vitro Assay for Measuring Neutrophil Serine Protease Activity Using a Fluorescent Reporter

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An In Vitro Assay for Neutrophil Serine Protease Activity Using a Fluorescent Reporter