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

Overview

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.

Protocol

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.

1. If spontaneous expectoration of sputum is not possible, induce sputum as described previously. Briefly, inhale 200 µg of the β-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.
2. Separate the mucus clumps from the saliva into a Petri dish with the help of a pipette tip.
3. 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.
4. 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.
5. 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.
6. Quench the reaction by adding the same volume of cold PBS (for example: 1 mL of cold PBS for each mL of 10% Sputolysin).
7. Mix by pipetting to obtain a homogenous solution.
8. Filter the mixture through a 100 µm nylon cell strainer into a 50 mL tube.
9. Repeat the filtration step through a 40 µm nylon cell strainer.
10. Centrifuge the solution for 10 min at 300 x g at 4 °C.
11. Transfer the supernatant fraction carefully into a fresh tube and store it on ice.
    NOTE: The supernatant fractions can be stored at -20 °C or -80 °C until further analysis.
12. Gently resuspend the cell pellet in 500 µL of cold PBS and place it on ice.
    ​NOTE: The cell fraction must be processed immediately.

2. Neutrophil serine protease activity measurement

NOTE: Here, different methods are introduced to quantify NSP'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.

1. Soluble NSP's activity quantification via fluorimeter or plate reader assay
   ​NOTE: Protease activity in soluble fractions of the sample can be detected with any instrument capable of fluorescence detection.
   1. Thaw enzymes on ice.
   2. 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.
   3. 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.
      1. To prepare the highest standard concentration (NE concentration: 33.9 nM), dilute 1 µL of NE (33.9 µM) in 999 µL of activation buffer.
      2. To prepare the second standard (NE concentration: 16.95 nM), mix 200 µL of the first dilution with 200 µL of activation buffer.
      3. Proceed accordingly to prepare the remaining 1:2 dilutions.
      4. 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.
   4. 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.
   5. Before preparing the samples for measurement, set up the instrument. Set the excitation wavelength for the NE Förster 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).
   6. Add 40 µL of samples, standard or blank into the wells of a black 96-well half area plate.
   7. To prepare the master mix containing the reporters (concentration of the reporter in the master mix: 10 µM), dilute the probe stock (1 mM in dimethylsulfoxide, DMSO) 1:100 in activation buffer. Prepare the needed master mix volume by multiplying 10 µL x the number of required plate wells. To reach the optimal final concentration (2 µM) for fluorescence measurement of NEmo-1 and sSAM reporters, add 10 µL of the master mix to each well (containing 40 µL 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.
   8. 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.
   9. 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.
   10. Calculate the D/A ratio mean and standard deviation of each sample.
   11. 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.

Materials

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