Simultaneous detection of cellular viability and interleukin-1β secretion from single cells by ELISpot

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-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.