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Methods Mol Biol. 2016;1417:75-88. doi: 10.1007/978-1-4939-3566-6_4.

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

Author information

1
Faculty of Life Sciences, University of Manchester, AV Hill Building, Oxford Road, Manchester, UK.
2
Singapore Immunology Network (SIgN), Agency for Science Technology and Research (A*STAR), Singapore, Singapore.
3
Faculty of Life Sciences, University of Manchester, AV Hill Building, Oxford Road, Manchester, UK. david.brough@manchester.ac.uk.

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

PMID:
27221482
DOI:
10.1007/978-1-4939-3566-6_4
[Indexed for MEDLINE]

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