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Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples
1Translational Lung Research Center Heidelberg (TLRC),German Center for Lung Research (DZL), 2Dept. of Translational Pulmonology,University of Heidelberg, 3Molecular Medicine Partnership Unit (MMPU), European Molecular Biology Laboratory (EMBL),University of Heidelberg, 4Faculty of Biosciences, Collaboration for Joint Ph.D. Degree between EMBL and Heidelberg University,University of Heidelberg, 5Dept. of Pediatric Pulmonology, Immunology and Critical Care Medicine,Charité – Universitätsmedizin Berlin, 6Berlin Institute of Health (BIH), 7German Center for Lung Research (DZL), Associated Partner Site, Berlin, 8Dept. of Chemical Physiology and Biochemistry,Oregon Health and Science University
Chapters
- 00:05Introduction
- 01:07Cell Isolation and Supernatant Separation
- 02:36Quantification of Soluble Neutrophil Serine Protease (NSP) Activity via Fluorimeter or Plate Reader Assay
- 04:13Membrane-bound NSPs Activity Measurement via Fluorescence Microscopy
- 05:30Membrane-bound NSPs Activity Measurement via Flow Cytometry
- 07:25Results: Representative Images and Quantification of Membrane-Bound NE Activity on Neutrophils Isolated from CF Patient Sputum
- 08:26Conclusion
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.
