Inflammasome and Caspase-1 Activity Characterization and Evaluation: An Imaging Flow Cytometer-Based Detection and...

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Inflammasome dysregulation is implicated in numerous inflammatory diseases.
• Current methods for evaluating inflammasome activity, such as microscopy and time of flight inflammasome evaluation (TOFIE), are time-consuming, have small sample sizes, and cannot visualize ASC specks or caspase-1 activity simultaneously.
• This limitation hinders colocalization studies of inflammasome components and enzymatic activity.

Purpose of the Study:

• To develop a rapid, high-throughput, single-cell imaging method for quantitative and qualitative characterization of inflammasome-associated structures (ASC specks) and caspase-1 activity.
• To overcome the limitations of existing methods in visualizing and analyzing inflammasome assembly and activity at the single-cell level.

Main Methods:

• Utilized the Amnis ImageStream^X instrument for fluorescence-based image analysis.
• Developed a method to quantitatively and qualitatively characterize the frequency, area, and cellular distribution of ASC specks and caspase-1 activity in mouse and human cells.
• Differentiated between singular perinuclear specks and false positives, unlike TOFIE.

Main Results:

• The new method successfully characterized ASC specks and caspase-1 activity in single cells.
• Demonstrated that NLRP3 reduces ASC speck size, and active caspase-1 further reduces it.
• Showed the capacity to simultaneously detect and quantify ASC specks and caspase-1 activity at both population and single-cell levels.

Conclusions:

• The developed imaging method is a powerful tool for visualizing and quantifying inflammasome assembly and activity.
• This technique enables detailed studies on the impact of mutations on inflammasome components and function.
• Offers a significant advancement over existing methods for inflammasome research in inflammatory diseases.