Simultaneous detection of cellular viability and interleukin-1beta secretion from single cells by ELISpot
Journal Title
Methods Mol Biol
Publication Type
Journal Article
Abstract
Cell death results in the breakdown of the plasma membrane, which can cause the release of cytosolic proteins. During caspase-1-mediated cell death, termed pyroptosis, pro-inflammatory mediators that lack canonical secretory signal sequences, such as interleukin-1beta (IL-1beta), are released into the extracellular environment. To define whether cell death is required for the release of IL-1beta, or if IL-1beta can be actively secreted from viable cells, we have developed a modified IL-1beta Enzyme-Linked ImmunoSpot (ELISpot) assay. This assay simultaneously detects cellular viability and IL-1beta release at the single-cell level, and is therefore useful to examine how cell death influences IL-1beta secretion under different experimental conditions. Cells expressing a surrogate viability marker, such as GFP, are plated onto cellulose filter plates coated with an IL-1beta capture antibody. This antibody immobilizes IL-1beta as it is released from cells, allowing detection of distinct IL-1beta "spots." Both GFP positive cells and IL-1beta spots are detected and quantified using an AID ELISpot Reader, and the captured images are overlaid. Therefore, cell viability and IL-1beta release from individual cells can be monitored visually. We have recently used this method to document how individual fibroblasts expressing activated caspase-1 can secrete IL-1beta in the absence of cell death. Adaptation of this assay to other experimental conditions may help to define the circumstances where cell death influences IL-1beta release and IL-1beta-driven inflammatory responses.
Publisher
Humana Press Inc.
WEHI Research Division(s)
Cell Signalling And Cell Death; Inflammation
PubMed ID
29177866
Rights Notice
Refer to copyright notice on published article.


Creation Date: 2018-01-15 09:30:07
Last Modified: 2018-02-14 03:52:03
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