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.
authors.affiliation: Faculty of Life Sciences, University of Manchester, AV Hill Building, Oxford Road, Manchester, UK.|Singapore Immunology Network (SIgN), Agency for Science Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Catherine
authors.initials: C
authors.lastname: Diamond
authors.affiliation: Faculty of Life Sciences, University of Manchester, AV Hill Building, Oxford Road, Manchester, UK.
authors.forename: James
authors.initials: J
authors.lastname: Bagnall
authors.affiliation: Faculty of Life Sciences, University of Manchester, AV Hill Building, Oxford Road, Manchester, UK.
authors.forename: David G
authors.initials: DG
authors.lastname: Spiller
authors.affiliation: Faculty of Life Sciences, University of Manchester, AV Hill Building, Oxford Road, Manchester, UK.
authors.forename: Michael R
authors.initials: MR
authors.lastname: White
authors.affiliation: Singapore Immunology Network (SIgN), Agency for Science Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Mortellaro
authors.affiliation: Faculty of Life Sciences, University of Manchester, AV Hill Building, Oxford Road, Manchester, UK.
authors.forename: Pawel
authors.initials: P
authors.lastname: Paszek
authors.affiliation: Faculty of Life Sciences, University of Manchester, AV Hill Building, Oxford Road, Manchester, UK. david.brough@manchester.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Brough
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D053583:Interleukin-1beta / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: C587672:IL1B protein, mouse; D053583:Interleukin-1beta
keywords: Confocal microscopy; IL-1β Venus; IL-1β secretion; Lentiviral transduction; Real-time single-cell imaging