A comprehensive three-dimensional assay to assess neutrophil defense against bacteria

Erinke van Grinsven, Pieter H. C. Leliefeld, Janesh Pillay, Corneli W. van Aalst, Nienke Vrisekoop, Leo Koenderman*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

7 Citations (Scopus)
35 Downloads (Pure)

Abstract

Neutrophil antibacterial capacity is measured in animal models and in vitro as an important indicator of neutrophil function. To be able to extrapolate their conclusions, in vitro experiments should mimic the in vivo situation. In vivo, antibacterial capacity depends on multiple steps of bacterial sensing, priming, chemotaxis, phagocytosis and intracellular killing. Therefore, we developed a simply executed assay that involves multiple steps in one assay. The neutrophils were incorporated into a three-dimensional matrix of fibrin fibers, in which they could freely migrate. The fibrin matrix provided a more physiological representation of tissue structure than a shaken suspension and extended ex vivo survival of neutrophils. Staphylococci endogenously producing GFP (Green Fluorescent Protein) provided a real-time quantification of the bacterial load without the need for lysing the fibrin matrix or counting of colony forming units on agar plates. The delay in bacterial outgrowth serves as a measure for the relative antibacterial capacity of the neutrophils. Additionally, neutrophil capacity could easily be measured high-throughput in a 96-wells format.

In this new assay we study neutrophil behavior in a physiologically relevant setting and explore many functions of the neutrophil in a single test. The functional capacity of neutrophils from different in vitro treatments or different donors can directly be compared.

Original languageEnglish
Pages (from-to)83-90
Number of pages8
JournalJournal of Immunological Methods
Volume462
DOIs
Publication statusPublished - Nov-2018
Externally publishedYes

Keywords

  • Neutrophil
  • Staphylococcus
  • Killing
  • Phagocytosis
  • in vitro survival
  • STAPHYLOCOCCUS-AUREUS
  • RESPIRATORY BURST
  • FIBRIN GELS
  • IN-VITRO
  • ACTIVATION
  • MICE
  • MIGRATION
  • GLYCOPROTEINS
  • INFLAMMATION
  • MECHANISMS

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