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

Published: January 31, 2024

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 me…

Açıklamalar

The authors have nothing to disclose.

Materials

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

Etiketler

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Bu Makaleden Alıntı Yapın
An In Vitro Assay for Measuring Neutrophil Serine Protease Activity Using a Fluorescent Reporter. J. Vis. Exp. (Pending Publication), e21902, doi: (2024).

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