Visualizing P2X7-Dependent Inflammasome Formation in Human Monocytes by Fluorescence Microscopy and Flow Cytometry

Abstract

One of the most prominent effects of P2X7 activation in myeloid cells is the induction of the assembly of the NLRP3 inflammasome, a central process controlling the secretion of pro-inflammatory cytokines of the IL-1 family such as IL-1β and IL-18. The ability to visualize inflammasome formation greatly facilitates research into the role of P2X7 in inflammation. In this chapter, a method to monitor the formation of the NLPR3 inflammasome in monocytes and other myeloid cells could be demonstrated. Following priming by lipopolysaccharide (LPS), P2X7 was stimulated by ATP to mediate inflammasome assembly. This causes cytosolically disperse ASC, a central component of the inflammasome, to aggregate into microscopically visible specks due to its recruitment to the inflammasome. Methods to monitor this change in the spatial distribution of ASC in human peripheral blood monocytes by flow cytometry and fluorescence microscopy are presented.