Simultaneous Detection of Cellular Viability and Interleukin-1β Secretion from Single Cells by ELISpot
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-1β (IL-1β), are released into the extracellular environment. To define whether cell death is required for the release of IL-1β, or if IL-1β can be actively secreted from viable cells, we have developed a modified IL-1β Enzyme-Linked ImmunoSpot (ELISpot) assay. This assay simultaneously detects cellular viability and IL-1β release at the single-cell level, and is therefore useful to examine how cell death influences IL-1β secretion under different experimental conditions. Cells expressing a surrogate viability marker, such as GFP, are plated onto cellulose filter plates coated with an IL-1β capture antibody. This antibody immobilizes IL-1β as it is released from cells, allowing detection of distinct IL-1β “spots.” Both GFP positive cells and IL-1β spots are detected and quantified using an AID ELISpot Reader, and the captured images are overlaid. Therefore, cell viability and IL-1β 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-1β 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-1β release and IL-1β-driven inflammatory responses.
KeywordsInterleukin-1β Inflammasome ELISpot Apoptosis Cell death Cytokine Caspase-1 Caspase-8 Pyroptosis Secretion
We thank Melinda Hardy and Dimitra Zotos for ELISpot protocols, and David Vaux and Kate Lawlor for scintillating discussions and advice. This work was supported by National Health and Medical Research Council (Canberra, Australia) Project grants (1051210), fellowships (JEV ; LML ); and operational infrastructure grants through the Australian Government IRISS and the Victorian State Government OIS (361646).
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