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

doi:10.1007/978-1-4939-3566-6_4

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^*

^*Corresponding author for this work

Faculty of Life Sciences
University of Manchester
Agency for Science, Technology and Research, Singapore

Research output: Chapter in Book or Report/Conference proceeding › Chapter › peer-review

1 Citation (Scopus)

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.

Original language	English
Title of host publication	Methods in Molecular Biology
Publisher	Humana Press Inc.
Pages	75-88
Number of pages	14
DOIs	https://doi.org/10.1007/978-1-4939-3566-6_4
Publication status	Published - 2016
Externally published	Yes

Publication series

Name	Methods in Molecular Biology
Volume	1417
ISSN (Print)	1064-3745

Keywords

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

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10.1007/978-1-4939-3566-6_4

Cite this

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title = "Investigating IL-1β secretion using real-time single-cell imaging",

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.",

keywords = "Confocal microscopy, IL-1β secretion, IL-1β Venus, Lentiviral transduction, Real-time single-cell imaging",

author = "Catherine Diamond and James Bagnall and Spiller, \{David G.\} and White, \{Michael R.\} and Alessandra Mortellaro and Pawel Paszek and David Brough",

note = "Funding Information: The authors are grateful to Prof Claire Bryant from the University of Cambridge for providing the immortalised BMDMs. P.P. holds a BBSRCBiotechnology and Biological Sciences Research Council David Phillips Research Fellowship (BB/I017976/1). This work was also supported by Medical Research Council Canada M.R.C. (MR/K015885/1) and Biotechnology and Biological Sciences Research Council (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. Publisher Copyright: {\textcopyright} Springer Science+Business Media New York 2016.",

year = "2016",

doi = "10.1007/978-1-4939-3566-6\_4",

language = "English",

series = "Methods in Molecular Biology",

publisher = "Humana Press Inc.",

pages = "75--88",

booktitle = "Methods in Molecular Biology",

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T1 - Investigating IL-1β secretion using real-time single-cell imaging

AU - Diamond, Catherine

AU - Bagnall, James

AU - Spiller, David G.

AU - White, Michael R.

AU - Mortellaro, Alessandra

AU - Paszek, Pawel

AU - Brough, David

N1 - Funding Information: The authors are grateful to Prof Claire Bryant from the University of Cambridge for providing the immortalised BMDMs. P.P. holds a BBSRCBiotechnology and Biological Sciences Research Council David Phillips Research Fellowship (BB/I017976/1). This work was also supported by Medical Research Council Canada M.R.C. (MR/K015885/1) and Biotechnology and Biological Sciences Research Council (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. Publisher Copyright: © Springer Science+Business Media New York 2016.

PY - 2016

Y1 - 2016

N2 - 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.

AB - 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.

KW - Confocal microscopy

KW - IL-1β secretion

KW - IL-1β Venus

KW - Lentiviral transduction

KW - Real-time single-cell imaging

UR - https://www.scopus.com/pages/publications/84990194868

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DO - 10.1007/978-1-4939-3566-6_4

M3 - Chapter

C2 - 27221482

AN - SCOPUS:84990194868

T3 - Methods in Molecular Biology

SP - 75

EP - 88

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -

Investigating IL-1β secretion using real-time single-cell imaging

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