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

Abstract

Source: Frey, D. L., et al. Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples. 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.

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

Materials

List of materials used in this article

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