Live Imaging of Pyroptosis in Primary Murine Macrophages

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2691; p. 139
• Main Authors: Holley, Caroline L, Schroder, Kate
• Format: Journal Article
• Language: English
• Published: United States 2023
• Subjects: Inflammasome; Inflammation; Pyroptosis; Live imaging; Microscopy; Macrophage
• ISSN: 1940-6029
• DOI: 10.1007/978-1-0716-3331-1_11

Inflammasomes are the ultimate weapon of the macrophage immune arsenal. Inflammasome signalling in macrophages triggers pyroptosis, a lytic cell death pathway that facilitates inflammation-driven pathogen clearance. Imaging-based approaches to investigating cell death have proven useful, revealing cellular remodelling events such as the generation of extracellular vesicles, and continuing to uncover important structural changes in cells involved in inflammatory signalling. Pyroptosis has proved extremely challenging to image, because its lytic nature is incompatible with many well-established imaging approaches employed for other, non-lytic pathways. The complexities of ectopically expressing fluorescent constructs in primary macrophages and the sensitivity of such proteins to drug-based probes compound this difficulty. We and others have demonstrated key differences in pyroptosis induced by canonical versus noncanonical inflammasomes that delineate functional differences between these signalling pathways. Here, we describe a live imaging approach to study and compare canonical versus noncanonical inflammasome signalling and pyroptotic architecture in primary murine macrophages.