Simultaneous Detection of Cellular Viability and Interleukin-1β Secretion from Single Cells by ELISpot
| Autor: | Lisa M Lindqvist, Stephanie A. Conos, James E Vince |
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| Přispěvatelé: | Conos, Stephanie A, Lindqvist, Lisa M, Vince, James E |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Programmed cell death biology interleukin-1β Chemistry pyroptosis ELISPOT ELISpot apoptosis caspase-1 Pyroptosis Caspase 1 caspase-8 Cell biology secretion 03 medical and health sciences cell death 030104 developmental biology inflammasome cytokine Extracellular biology.protein Secretion Viability assay Antibody |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493975181 |
| DOI: | 10.1007/978-1-4939-7519-8_15 |
| Popis: | 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. Refereed/Peer-reviewed |
| Databáze: | OpenAIRE |
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