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

Area of Science:

• Immunology
• Cell Biology

Background:

• P2X7 receptor activation in myeloid cells triggers NLRP3 inflammasome assembly.
• Inflammasome activation controls the release of pro-inflammatory cytokines, including IL-1β and IL-18.
• Visualizing inflammasome formation is crucial for understanding P2X7's role in inflammatory processes.

Purpose of the Study:

• To present a method for monitoring NLRP3 inflammasome formation in monocytes and myeloid cells.
• To demonstrate how P2X7 stimulation by ATP, after lipopolysaccharide (LPS) priming, induces inflammasome assembly.
• To facilitate research on P2X7-mediated inflammation by enabling visualization of inflammasome dynamics.

Main Methods:

• Priming myeloid cells with lipopolysaccharide (LPS).
• Stimulating P2X7 receptors with Adenosine triphosphate (ATP) to induce inflammasome assembly.
• Monitoring the spatial redistribution of ASC (pyrin domain-containing protein 3) from a diffuse cytosolic state to visible specks using flow cytometry and fluorescence microscopy.

Main Results:

• P2X7 stimulation leads to the aggregation of ASC into detectable specks, indicating inflammasome assembly.
• The presented method allows for the visualization of inflammasome formation in human peripheral blood monocytes.
• Changes in ASC localization can be quantified using flow cytometry and observed via fluorescence microscopy.

Conclusions:

• The described method effectively visualizes NLRP3 inflammasome assembly in myeloid cells.
• This technique enhances the study of P2X7 receptor function and its role in inflammation.
• Visualizing ASC speck formation provides a measurable readout for inflammasome activation.