This project was supported by grants from the German Ministry for Education and Research (FKZ 82DZL004A1 to M.A.M) and the German Research Foundation (SFB-TR84TP B08 to M.A.M). Work described in this manuscript was supported by the German Center of Lung Research (DZL) and the EMBL Heidelberg through a PhD fellowship for M.G. We thank J. Schatterny, S. Butz and H. Scheuermann for expert technical assistance.

<ol>
	<li>Korkmaz, B., Moreau, T., Gauthier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+elastase,+proteinase+3+and+cathepsin+G:+Physicochemical+properties,+activity+and+physiopathological+functions.">Neutrophil elastase, proteinase 3 and cathepsin G: Physicochemical properties, activity and physiopathological functions.</a> Biochimie. 90 (2), 227-242 (2008).</li><li>Sheshachalam, A., Srivastava, N., Mitchell, T., Lacy, P., Eitzen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granule+Protein+Processing+and+Regulated+Secretion+in+Neutrophils.">Granule Protein Processing and Regulated Secretion in Neutrophils.</a> Frontiers in Immunology. 5, 448 (2014).</li><li>Pham, C. T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+serine+proteases:+Specific+regulators+of+inflammation.">Neutrophil serine proteases: Specific regulators of inflammation.</a> Nature Reviews Immunology. 6 (7), 541-550 (2006).</li><li>Gehrig, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+neutrophil+elastase+reduces+inflammation,+mucus+hypersecretion,+and+emphysema,+but+not+mucus+obstruction,+in+mice+with+cystic+fibrosislike+lung+disease.">Lack of neutrophil elastase reduces inflammation, mucus hypersecretion, and emphysema, but not mucus obstruction, in mice with cystic fibrosislike lung disease.</a> American Journal of Respiratory and Critical Care Medicine. 189 (9), 1082-1092 (2014).</li><li>McKelvey, M. C., Weldon, S., McAuley, D. F., Mall, M. A., Taggart, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+proteases+in+cystic+fibrosis+lung+disease+paradigms,+progress,+and+potential.">Targeting proteases in cystic fibrosis lung disease paradigms, progress, and potential.</a> American Journal of Respiratory and Critical Care Medicine. 201 (2), 141-147 (2020).</li><li>Clancy, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Neutrophil+Proteases+Are+Efficient+Regulators+of+IL-1,+IL-33,+and+IL-36+Cytokine+Activity+but+Poor+Effectors+of+Microbial+Killing.">Extracellular Neutrophil Proteases Are Efficient Regulators of IL-1, IL-33, and IL-36 Cytokine Activity but Poor Effectors of Microbial Killing.</a> Cell Reports. 22 (11), 2937-2950 (2018).</li><li>Giacalone, V. D., Margaroli, C., Mall, M. A., Tirouvanziam, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adaptations+upon+recruitment+to+the+lung:+New+concepts+and+implications+for+homeostasis+and+disease.">Neutrophil adaptations upon recruitment to the lung: New concepts and implications for homeostasis and disease.</a> International Journal of Molecular Sciences. 21 (3), 1-21 (2020).</li><li>Sly, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+Factors+for+Bronchiectasis+in+Children+with+Cystic+Fibrosis.">Risk Factors for Bronchiectasis in Children with Cystic Fibrosis.</a> New England Journal of Medicine. 368 (21), 1963-1970 (2013).</li><li>Owen, C. A., Campbell, M. A., Sannes, P. L., Boukedes, S. S., Campbell, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface-bound+elastase+and+cathepsin+G+on+human+neutrophils:+A+novel,+non-oxidative+mechanism+by+which+neutrophils+focus+and+preserve+catalytic+activity+of+serine+proteinases.">Cell surface-bound elastase and cathepsin G on human neutrophils: A novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases.</a> Journal of Cell Biology. 131 (3), 775-789 (1995).</li><li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Extracellular+Traps+Kill+Bacteria.">Neutrophil Extracellular Traps Kill Bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li><li>Margaroli, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastase+Exocytosis+by+Airway+Neutrophils+Associates+with+Early+Lung+Damage+in+Cystic+Fibrosis+Children.">Elastase Exocytosis by Airway Neutrophils Associates with Early Lung Damage in Cystic Fibrosis Children.</a> American Journal of Respiratory and Critical Care Medicine. , (2018).</li><li>Owen, C., Campbell, M. A., Sannes, P. L., Boukedes, S. S., Carolina, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface-bound+elastase+and+cathepsin+G+on+human+neutrophils:+a+novel,+non-oxidative+mechanism+by+which+neutrophils+focus+and+preserve+catalytic+activity+of+serine+proteinases.">Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases.</a> The Journal of cell biology. 131 (3), 775-789 (1995).</li><li>Garland, M., Yim, J. J., Bogyo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Bright+Future+for+Precision+Medicine:+Advances+in+Fluorescent+Chemical+Probe+Design+and+Their+Clinical+Application.">A Bright Future for Precision Medicine: Advances in Fluorescent Chemical Probe Design and Their Clinical Application.</a> Cell chemical biology. 23 (1), 122-136 (2016).</li><li>Gehrig, S., Mall, M. A., Schultz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+resolved+monitoring+of+neutrophil+elastase+activity+with+ratiometric+fluorescent+reporters.">Spatially resolved monitoring of neutrophil elastase activity with ratiometric fluorescent reporters.</a> Angewandte Chemie - International Edition. 51 (25), 6258-6261 (2012).</li><li>Guerra, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cathepsin+G+Activity+as+a+New+Marker+for+Detecting+Airway+Inflammation+by+Microscopy+and+Flow+Cytometry.">Cathepsin G Activity as a New Marker for Detecting Airway Inflammation by Microscopy and Flow Cytometry.</a> ACS Central Science. 5 (3), 539-548 (2019).</li><li>Hu, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+mouse+tumors+by+a+lipidated+cathepsin+S+substrate.">In vivo imaging of mouse tumors by a lipidated cathepsin S substrate.</a> Angewandte Chemie - International Edition. 53 (29), 7669-7673 (2014).</li><li>Korkmaz, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+elastase,+proteinase+3+and+cathepsin+G+activities+at+the+surface+of+human+neutrophils+with+fluorescence+resonance+energy+transfer+substrates.">Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates.</a> Nature Protocols. 3 (6), 991-1000 (2008).</li><li>Craven, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-silent+FRET+Sensor+Enables+Live+Cell+Imaging+and+Flow+Cytometric+Stratification+of+Intracellular+Serine+Protease+Activity+in+Neutrophils.">Super-silent FRET Sensor Enables Live Cell Imaging and Flow Cytometric Stratification of Intracellular Serine Protease Activity in Neutrophils.</a> Scientific Reports. 8 (1), 13490 (2018).</li><li>Mu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small-molecule+fret+reporter+for+the+real-time+visualization+of+cell-surface+proteolytic+enzyme+functions.">A small-molecule fret reporter for the real-time visualization of cell-surface proteolytic enzyme functions.</a> Angewandte Chemie - International Edition. 53 (52), 14357-14362 (2014).</li><li>Hagner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+method+for+rapid+and+dynamic+quantification+of+elastase+activity+on+sputum+neutrophils+from+patients+with+cystic+fibrosis+using+flow+cytometry.">New method for rapid and dynamic quantification of elastase activity on sputum neutrophils from patients with cystic fibrosis using flow cytometry.</a> European Respiratory Journal. 55 (4), 1902355 (2020).</li><li>Dittrich, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastase+activity+on+sputum+neutrophils+correlates+with+severity+of+lung+disease+in+cystic+fibrosis.">Elastase activity on sputum neutrophils correlates with severity of lung disease in cystic fibrosis.</a> European Respiratory Journal. , 1701910 (2018).</li><li>Cobos-Correa, A., Trojanek, J. B., Diemer, S., Mall, M. A., Schultz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-bound+FRET+probe+visualizes+MMP12+activity+in+pulmonary+inflammation.">Membrane-bound FRET probe visualizes MMP12 activity in pulmonary inflammation.</a> Nature Chemical Biology. 5 (9), 628-630 (2009).</li><li>Hu, H. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET-based+and+other+fluorescent+proteinase+probes.">FRET-based and other fluorescent proteinase probes.</a> Biotechnology Journal. 9 (2), 266-281 (2014).</li><li>Trojanek, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+mucus+obstruction+triggers+macrophage+activation+and+matrix+metalloproteinase+12-dependent+emphysema.">Airway mucus obstruction triggers macrophage activation and matrix metalloproteinase 12-dependent emphysema.</a> American Journal of Respiratory Cell and Molecular Biology. 51 (5), 709-720 (2014).</li><li>Wagner, C. J., Schultz, C., Mall, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+elastase+and+matrix+metalloproteinase+12+in+cystic+fibrosis+lung+disease.">Neutrophil elastase and matrix metalloproteinase 12 in cystic fibrosis lung disease.</a> Molecular and Cellular Pediatrics. 3 (1), 25 (2016).</li><li>Gaggar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+matrix+metalloproteinases+in+cystic+fibrosis+lung+disease.">The role of matrix metalloproteinases in cystic fibrosis lung disease.</a> The European respiratory journal. 38 (3), 721-727 (2011).</li><li>Guerra, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease+FRET+Reporters+Targeting+Neutrophil+Extracellular+Traps.">Protease FRET Reporters Targeting Neutrophil Extracellular Traps.</a> Journal of the American Chemical Society. 142 (48), 20299-20305 (2020).</li><li>Middleton, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elexacaftor-Tezacaftor-Ivacaftor+for+Cystic+Fibrosis+with+a+Single+Phe508del+Allele.">Elexacaftor-Tezacaftor-Ivacaftor for Cystic Fibrosis with a Single Phe508del Allele.</a> New England Journal of Medicine. 381 (19), 1809-1819 (2019).</li></ol>

The authors declare no conflict of interests.

The reported protocols explain different approaches to quantify the activity of neutrophil elastase and cathepsin G in human sputum samples. Critical points for a successful enzyme activity measurement are the i) precise timing and standardization of the operative procedure and ii) the use of reliable negative and positive controls. If these conditions are met, the described methods are not limited to sputum but can also be easily adapted to the analysis of protease activity in blood, bronchoalveolar lavage fluids and tissue sections or homogenates.
Each of the three techniques has its strengths and limitations, which often complement each other. For example, flow cytometry allows for the rapid analysis of rare cell populations as well as cell phenotyping but lacks spatial resolution information, which can be achieved by microscopy. Instead, plate reader measurements permit for the parallel assessment of several samples or conditions in a high-throughput fashion. Since fresh sputum cells cannot be frozen and stored, the three methods require that samples must be processed rapidly after expectoration. This limits the flexibility or the throughput of the membrane-bound activity measurements. The development of a flow cytometry protocol that allows to fix cells after probe addition and enzymatic cleavage would open to the parallel measurement of a higher number of tubes. Moreover, particular attention should be paid to the handling and storage of the FRET probes. In fact, some aminoacids present in the peptide substrate, such as methionine, undergo oxidation which leads to decreased reporter sensitivity. To increase the reporter&#39;s shelf life (estimated of&#160;about three months at 20 &#176;C), they can be stored in small volume aliquots (1-2 &#181;L) under inert gas such as Nitrogen or Argon.
In CF and other chronic inflammatory lung diseases it is important to detect the inflammation as early as possible, and reliable biomarkers have the potential to achieve such a goal. The possibility to detect surface-bound NSPs activity, which has been shown to be harmful for the surrounding tissue, also in conditions when there is no or little free NE activity, adds another level of valuable information, which can be hardly achieved by means of other existing methods4,11.
The reporters can be used to study the link of membrane-bound associated NSP activity with severity and progression of lung disease, especially at its early-onset. The methods can be utilized to monitor treatment efficacy (e.g., anti-inflammatory treatments or highly effective CFTR modulators and potentiators28) and investigate the resulting dampening of neutrophil-driven inflammation. In addition, the protocols are based on non-invasive sample procedures which carry very low risk for the patient and, therefore, can be used on a very broad scale and open the doors to numerous exciting applications.

Neutrophil elastase (NE), cathepsin G (CG), proteinase 3 (PR3) and neutrophil serine protease 4 (NSP4) are the four neutrophil serine proteases (NSPs)1. They are stored, together with myeloperoxidase, within neutrophil primary or azurophilic granules. Due to their elevated proteolytic content, the secretion of primary granules is tightly regulated and neutrophils have to be sequentially challenged with priming and activating stimuli2.
Inside the phagolysosome, NSPs function as intracellular bactericidal agents3. When secreted, NSPs become strong mediators of inflammation: they cleave cytokines and surface receptors, activating parallel pro-inflammatory pathways3. Importantly, inflammatory conditions feature an uncontrolled NSPs secretion. For example, within inflamed airways, excessive NE activity causes mucus hypersecretion, goblet cells metaplasia, CFTR inactivation and extracellular matrix remodeling4,5. Cathepsin G participates in inflammation as well: it specifically cleaves and activates two components of the IL-1 family, IL-36&#945; and IL-36&#946;6. In concert with NE, CG cleaves protease-activated receptors on the airway epithelium and also activates TNF-&#945; and IL-1&#946;.
Endogenous anti-proteases such as alpha-1-antitrypsin, alpha-1-antichymotrypsin and the secretory leukocyte protease inhibitor regulate neutrophil elastase and cathepsin G activity5. However, over the course of lung disease progression, the continuous secretion of proteases exceeds stoichiometrically the anti-protease shield, leading to non-resolving neutrophilia in the airways, inflammation worsening and tissue damage5,7. Although NE concentration and activity in soluble fractions of patient airways has been shown to be a promising biomarker of disease severity8, NE and CG also associate to the neutrophil plasma membrane and to extracellular DNA via electrostatic interactions9,10 where they become less accessible to anti-proteases. Importantly, preclinical studies defined a scenario where cell surface-associated protease activity appears earlier and/or independently of its soluble counterpart4,11. In fact, to become detectable, free protease activity first needs to overwhelm the anti-protease shield. Instead, at the cell surface, membrane-bound protease activity remains at least partially intact due to the inaccessibility of large inhibitors to the cell plasma membrane12. Such complex protease behavior has important consequences on neutrophil-mediated inflammation onset and propagation, and therefore needs to be investigated with precise and informative tools.
Over the years, F&#246;rster resonance energy transfer (FRET)-based probes found numerous biomedical applications as tools that efficiently and rapidly assess a specific protease activity in human samples13. To function, protease reporters are composed of a recognition motif (i.e., a peptide), which is recognized by the target enzyme and rely on FRET, a physical process where, upon excitation, a donor fluorophore transfers energy to an acceptor molecule. The processing operated by the enzyme on the reporter, namely the cleavage of the recognition part, results in the acceptor to diffuse away from the donor: the enzyme activity is therefore measured as a time-dependent change in the donor over the acceptor fluorescence. Such read-out is self-normalizing and ratiometric, hence only marginally affected by environmental conditions such as pH and local probe concentration. NEmo-114 and sSAM15 are FRET probes that report specifically on NE and CG activity, respectively. However, such reporters do not localize specifically to any cellular compartment, therefore they are employed to monitor the protease activity present in human fluids. In order to monitor protease activity in a spatially localized fashion, we and others developed FRET probes that associate to subcellular components via molecular tags14,15,16,17,18,19. Such a synthetic strategy allowed the development of NEmo-2 and mSAM, two FRET probes equipped with lipid anchors that localize to the plasma membrane. These reporters fueled a deeper understanding of NE and CG proteases in cystic fibrosis and chronic obstructive pulmonary diseases14,15.
Here, detailed protocols are provided for the visualization and quantification of soluble and membrane-bound NE and CG activities in human sputum by means of NEmo and SAM series of FRET probes. To address diverse aspects of NSPs pathophysiology and provide an array of methods that can be employed according to the user-specific need, the analysis via fluorescence spectroscopy, fluorescence microscopy and flow cytometry are shown.

<table><tbody><tr><td>100 &amp;micro;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 &amp;micro;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>7-AAD, viability dye</td><td>Bio Legend</td><td>420404</td><td>5 &amp;micro;L/100 &amp;micro;L</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>Countig chambers improved Neubauer</td><td>Glaswarenfabrik Karl Hecht GmbH &amp;amp; Co KG</td><td>40442</td><td></td></tr><tr><td>coverslips &amp;Oslash; 25mm</td><td>Thermo Fisher Scientific</td><td>MENZCB00250RA003</td><td></td></tr><tr><td>Cytospin 4</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>DRAQ5 (nuclear stain)</td><td>BioStatus Limited</td><td>DR50050</td><td>1:10000</td></tr><tr><td>FACSDiva software, v8.0.1</td><td>BD Bioscience</td><td></td><td></td></tr><tr><td>FcBlock</td><td>BD Bioscience</td><td>564219</td><td></td></tr><tr><td>Fiji (Fiji Is Just ImageJ)</td><td>fiji.sc</td><td></td><td></td></tr><tr><td>Flow Jo software, v10</td><td>TreeStar</td><td></td><td></td></tr><tr><td>FluoQ Plugin, v3-97</td><td></td><td></td><td></td></tr><tr><td>Heraeus Megafuge 16R</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Human Sputum Leucocyte Elastase</td><td>Elastin Products Company</td><td>SE563</td><td></td></tr><tr><td>Leica SP8 confocal microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Mini Rock-Shaker</td><td>PEQLAB Biotechnologie GmbH</td><td>MR-1</td><td></td></tr><tr><td>mouse anti-human CD14, Pe-Cy7, clone M5E2</td><td>BD Bioscience</td><td>557742 Lot:8221983</td><td>1:50</td></tr><tr><td>mouse anti-human CD16, AF700, clone 3G8</td><td>BD Bioscience</td><td>557820 Lot:8208791</td><td>1:50</td></tr><tr><td>mouse anti-human CD45, APC-Cy7, clone 2D1</td><td>BD Bioscience</td><td>557833 Lot:8059688</td><td>1:33</td></tr><tr><td>mouse anti-human CD66b, PE/Dazzel 594, clone G10F5</td><td>BioLegend</td><td>305122 Lot:B241921</td><td>1:50</td></tr><tr><td>mSAM</td><td>in house</td><td></td><td>2 mM</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><tr><td>SuperFrost Plus Adhesion slides</td><td>Thermo Fisher Scientific</td><td>10149870</td><td></td></tr><tr><td>Trypan Blue solution</td><td>Sigma-Aldrich</td><td>T8154</td><td></td></tr></tbody></table>

monitoring neutrophil elastase and cathepsin g activity in human sputum samples

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical challenge. Among the ~600 proteases encoded by the human genome, neutrophil serine proteases (NSPs) are thoroughly investigated for their involvement in the onset and progression of inflammatory conditions including respiratory diseases. Uniquely, secreted NSPs not only diffuse within extracellular fluids but also localize to plasma membranes. During neutrophil extracellular trap (NETs) formation, NSPs become an integral part of the secreted chromatin. Such complex behavior renders the understanding of NSPs pathophysiology a challenging task. Here, detailed protocols are shown to visualize, quantify and discriminate free and membrane-bound neutrophil elastase (NE) and cathepsin G (CG) activities in sputum samples. NE and CG are NSPs whose activities have pleiotropic roles in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD): they promote tissue remodeling, regulate downstream immune responses and correlate with lung disease severity. The protocols show how to separate fluid and cellular fraction, as well as the isolation of neutrophils from human sputum for enzymatic activity quantification via small-molecule F&#246;rster resonance energy transfer-based (FRET) reporters. To gather specific insights into the relative role of NE and CG activities, a FRET readout can be measured by different technologies: i) in vitro plate reader measurements allow for high-throughput and bulk detection of protease activity; ii) confocal microscopy spatiotemporally resolves membrane-bound activity at the cell surface; iii) small-molecule FRET flow cytometry enables for the rapid evaluation of anti-inflammatory treatments via single-cell protease activity quantification and phenotyping. The implementation of such methods opens the doors to explore NSPs pathobiology and their potential as biomarkers of disease severity for CF and COPD. Given their standardization potential, their robust readout and simplicity of transfer, the described techniques are immediately shareable for implementation across research and diagnostic laboratories.

The results shown in Figure 1a illustrate a representative microscopy dataset. The nuclear signal is used to identify neutrophils by their characteristic segmented nuclei. The region of interest (ROI) is selected manually (dashed line in Figure 1a). The D/A ratio image is calculated by dividing the intensities of the donor channel by the intensities of the acceptor channel on a pixel-by-pixel basis. In the last step the average D/A ratio per cell (ROI) is calculated. In Figure 1b each dot represents the mean of one ROI (neutrophil). It is recommended to image and evaluate about 100 cells per condition.
A representative flow cytometry gating strategy is shown in Figure 2a. Such gating allows to discriminate and study sputum neutrophils. To avoid fluorescence spillover or compensation artifacts, it is recommended to dedicate a laser line (e.g., blue laser) to the FRET probe fluorescence detection. Flow cytometry fluorescence compensation should be performed for antibodies and not for the FRET probe. Figure 2b depicts the MFI distribution at 0 and 10 min after reporter addition. Each row in Figure 2c indicates the mean donor and acceptor MFIs values for 1000 sputum neutrophils. The D/A ratio is calculated by dividing the donor and acceptor MFIs. The time course in Figure 2c on the right side shows the progression of the measurement: after a rapid initial increase, the D/A ratio reaches a plateau, according to the activity of the membrane-bound enzyme.

Watch this Scientific Journal Video about Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples at JoVE.com

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies.

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical ...

monitoring-neutrophil-elastase-cathepsin-g-activity-human-sputum

Research

JoVE Journal

Immunology and Infection

15.7K Views.  German Center for Lung Research (DZL). Onze FRET-sonde en protocollen maken de snelle en gevoelige kwantificering van vrije en oppervlaktegebonden elastase- en cathespin G-activiteit in sputum mogelijk bij patiënten met neutrofiele luchtwegaandoeningen. Deze methoden maken het mogelijk om verschillende aspecten van proteasen pathofysiologie te verkennen. Plaatlezermetingen maken grote screenings mogelijk.Confocale microscopie visualiseert enzymactiviteit met subcellulaire oplossing. Terwijl flow cytometrie, maakt eencellig...

Video: Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Cancer Research

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient&#39;s immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Medicine

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition.
The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (&#8805; 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

We present a protocol for a modified sandwich enzyme-linked immunosorbent assay technique to quantitatively measure two components of neutrophil extracellular trap remnants, myeloperoxidase conjugated-DNA and neutrophil elastase conjugated-DNA complexes,derived from activated neutrophils.

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...

Quantifying NET Formation Using Dual-Dye Live Cell Analysis

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

Author Spotlight: Quantifying Neutrophil Extracellular Traps in Disease and Drug Screening Using Dual-Color Live-Cell Imaging

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

Real-Time Microscopy for Studying NETosis Kinetics and Screening Inhibitors

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in a process referred to as NETosis. However, in the past two decades, it has become clear that the accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases. Therefore, interest in the development of NETosis antagonists has risen. Variable and non-standardized methods to detect and analyze NETosis were developed concomitantly, each with its own advantages and limitations. Here, we describe a real-time microscopy method for the quantification of human NET release, allowing to study NETosis as well as NET inhibition in a high-throughput manner. The surface area-based semi-automated analysis recognizes NETs and distinguishes them from non-netting activated neutrophils. We demonstrate that the non-physiological NETosis inducers, calcium ionophore and phorbol-12-myristate-13-acetate (PMA), trigger the release of NETs with different characteristics and kinetics. Furthermore, we show that this approach allows studying NET release in response to disease-relevant stimuli, including immune complexes, N-Formylmethionine-leucyl-phenylalanine (fMLF), monosodium urate crystals, and calcium pyrophosphate crystals. To exemplify the utility of this method to study NETosis antagonists, we used CIT-013, a first-in-class monoclonal antibody inhibitor of NET release. CIT-013 targets citrullinated histone H2A and H4 and efficiently inhibits NET release with an IC50 of 4.6 nM. Other anti-histone antibodies tested lacked this NETosis-inhibitory capacity. Altogether, we demonstrate that this protocol enables specific, reliable, and reproducible high-throughput quantification of NETs, enhancing the study of NET release characteristics, kinetics, and pharmacology of NETosis antagonists.

This defined protocol describes a real-time high-throughput microscopy approach to visualize and quantify human neutrophil extracellular trap (NET) release in vitro. The reproducible method allows investigation of the characteristics and kinetics of NET release upon stimulation with distinct NETosis inducers and enables assessment of the pharmacology of NETosis antagonists.

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in ...

Biology

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/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.

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

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video