Simultaneous detection of cellular viability and interleukin-1beta secretion from single cells by ELISpot
- Journal Title
- Methods Mol Biol
- Publication Type
- Journal Article
- 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.
- Humana Press Inc.
- WEHI Research Division(s)
- Cell Signalling And Cell Death; Inflammation
- PubMed ID
- Publisher's Version
- Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2018-01-15 09:30:07Last Modified: 2018-02-14 03:52:03