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
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).