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A novel flow cytometric method to assess inflammasome formation
David P Sester1, Sara J Thygesen2, Vitaliya Sagulenko2
1School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia; katryn.stacey@uq.edu.au d.sester@uq.edu.au.
Journal of Immunology (Baltimore, Md. : 1950)
|November 19, 2014
Summary
We developed a new assay to detect inflammasome formation by tracking the movement of the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). This method quantifies inflammasome assembly in mixed cell populations ex vivo.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Inflammasomes are crucial protein complexes mediating inflammatory responses and cell death.
- Inflammasome assembly involves the aggregation of the ASC adapter protein into a distinct cellular speck.
- Quantifying inflammasome formation in mixed cell populations ex vivo presents a significant technical challenge.
Purpose of the Study:
- To develop a novel flow cytometry-based assay for detecting and quantifying inflammasome formation.
- To characterize the assay's ability to analyze ASC relocalization within cells.
- To establish a sensitive method for studying inflammasome assembly dynamics and molecular interactions.
Main Methods:
- Development of a flow cytometric assay termed time of flight inflammasome evaluation (TIE).
- Detection of inflammasome formation via changes in ASC protein distribution (pulse width/height) using flow cytometry.
- Application of the TIE assay to quantify inflammasome assembly in mixed cell populations ex vivo.
Main Results:
- The TIE assay successfully detects the dramatic relocalization of ASC into a single speck during inflammasome formation.
- ASC transit into the speck is identified by characteristic changes in fluorescence pulse characteristics (decreased width or increased height).
- The assay enables quantification of native inflammasome formation in specific cell subsets within mixed populations.
Conclusions:
- The TIE assay provides a rapid, sensitive, and quantitative method for assessing inflammasome formation ex vivo.
- This assay facilitates the investigation of molecular interactions critical for inflammasome assembly through reconstitution experiments.
- The developed technique offers a valuable tool for immunological and cell biology research.
