Detection of Inflammasome Activation via Fluorescence Microscopy
Transcript
To detect NOD-like receptor P3, or NLRP3, inflammasome activation, take a multi-well plate containing activated macrophages expressing NLRP3 proteins.
Treat the cells with media containing ionophores and glycine. Ionophores induce potassium ion efflux, activating and oligomerizing NLRP3 proteins. Oligomerized NLRP3 recruits adaptor proteins, facilitating the binding of pro-caspase-1, forming the NLRP3 inflammasome complex.
On the inflammasome, proximity triggers pro-caspase-1 auto-cleavage, releasing active caspases, initiating pyroptosis and nuclear condensation. Additionally, glycine stabilizes cell structure, preventing cell lysis.
Treat macrophages with fluorescent reporter probes containing a caspase-1 binding group and a fluorophore linked via an amino acid sequence. The activated caspase-1 recognizes the probe's amino acid sequence and binds to its caspase-binding group, enabling its visualization.
Fix the cells and overlay them with fluorescent dye to stain the nuclei. Image under a fluorescence microscope.
Count macrophages with blue condensed nuclei and green fluorescent foci of activated caspase-1, suggesting NLRP3 inflammasome activation.
