Protocol | Published:

Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates

Nature Protocols volume 3, pages 9911000 (2008) | Download Citation

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Abstract

The neutrophil serine proteases (NSPs) elastase, proteinase 3 and cathepsin G are multifunctional proteases involved in pathogen destruction and the modulation of inflammatory processes. A fraction of secreted NSPs remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities on neutrophil surfaces using highly sensitive Abz-peptidyl-EDDnp fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. We describe FRET substrate synthesis, neutrophil purification and handling, and kinetic experiments on quiescent and activated cells. These are used to measure subnanomolar concentrations of membrane-bound or free NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids like expectorations and bronchoalveolar lavages. The whole procedure, including neutrophil purification and kinetic measurements, can be done in 4–5 h and should not be longer because of the lifetime of neutrophils. Using this protocol will help identify the contributions of individual NSPs to the development of inflammatory diseases and may reveal these proteases to be targets for therapeutic inhibitors.

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Acknowledgements

B.K. was funded by the Alexander von Humboldt Foundation and the German Research Council. This work was supported in France by Vaincre la Mucoviscidose and in Brazil by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-PADCT). The authors thank Owen Parkes for editing the English text.

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Affiliations

  1. Department of Neuroimmunology, Max-Planck Institute of Neurobiology, Planegg-Martinsried D-82152, Germany.

    • Brice Korkmaz
  2. INSERM U618 'Protéases et Vectorisation Pulmonaires' Faculty of medicine, 10 Bd Tonnellé, Tours 37032, France.

    • Brice Korkmaz
    • , Sylvie Attucci
    • , Timofey Kalupov
    •  & Francis Gauthier
  3. Université François Rabelais, 10 Bd Tonnellé, Tours 37032, France.

    • Sylvie Attucci
    • , Timofey Kalupov
    •  & Francis Gauthier
  4. Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal, São Paulo 04044-20, Brazil.

    • Maria Aparecida Juliano
    •  & Luiz Juliano
  5. INSERM U 921 'Nutrition, Croissance et Cancer' 10 Bd Tonnellé, Tours 37032, France.

    • Marie-Lise Jourdan

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Correspondence to Francis Gauthier.

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https://doi.org/10.1038/nprot.2008.63

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