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Investigating IL-1β Secretion Using Real-Time Single-Cell Imaging

  • Catherine Diamond
  • James Bagnall
  • David G. Spiller
  • Michael R. White
  • Alessandra Mortellaro
  • Pawel Paszek
  • David Brough
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1417)

Abstract

The pro-inflammatory cytokine interleukin (IL)-1β is an important mediator of the inflammatory response. In order to perform its role in the inflammatory cascade, IL-1β must be secreted from the cell, yet it lacks a signal peptide that is required for conventional secretion, and the exact mechanism of release remains undefined. Conventional biochemical methods have limited the investigation into the processes involved in IL-1β secretion to population dynamics, yet heterogeneity between cells has been observed at a single-cell level. Here, greater sensitivity is achieved with the use of a newly developed vector that codes for a fluorescently labelled version of IL-1β. Combining this with real-time single-cell confocal microscopy using the methods described here, we have developed an effective protocol for investigating the mechanisms of IL-1β secretion and the testing of the hypothesis that IL-1β secretion requires membrane permeabilisation.

Key words

Confocal microscopy IL-1β secretion Lentiviral transduction IL-1β Venus Real-time single-cell imaging 

Notes

Acknowledgements

The authors are grateful to Prof Claire Bryant from the University of Cambridge for providing the immortalised BMDMs. P.P. holds a BBSRC David Phillips Research Fellowship (BB/I017976/1). This work was also supported by M.R.C. (MR/K015885/1) and BBSRC (BB/K003097/1). The work leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2012-2017) under grant agreement n° 305564.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Catherine Diamond
    • 1
    • 2
  • James Bagnall
    • 1
  • David G. Spiller
    • 1
  • Michael R. White
    • 1
  • Alessandra Mortellaro
    • 2
  • Pawel Paszek
    • 1
  • David Brough
    • 1
  1. 1.Faculty of Life SciencesUniversity of ManchesterManchesterUK
  2. 2.Singapore Immunology Network (SIgN), Agency for Science Technology and Research (A*STAR)SingaporeSingapore

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