Comparative Analysis of Canonical Inflammasome Activation by Flow Cytometry, Imaging Flow Cytometry and High-Content...

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Inflammasome activation is implicated in various diseases, necessitating reliable detection methods.
• ASC speck+ cell detection via flow cytometry is crucial for studying inflammasome activation in patient samples.
• Standardization and inter-device comparability are critical challenges in flow cytometric analysis of inflammasome activation.

Purpose of the Study:

• To evaluate the performance of different flow cytometers in detecting ASC speck+ cells.
• To compare flow cytometry with fluorescence microscopy, imaging flow cytometry, and high-content imaging (HCI) for inflammasome activation assessment.
• To identify optimal methods for standardized and reliable detection of inflammasome activation.

Main Methods:

• Analysis of stimulated and unstimulated peripheral blood mononuclear cells (PBMCs) from healthy donors.
• Utilized seven different flow cytometers for ASC speck+ cell detection.
• Employed fluorescence microscopy, imaging flow cytometry, and high-content imaging (HCI) for comparative analysis.

Main Results:

• Significant variations were observed in the ability of different flow cytometers to detect ASC speck+ cells.
• Flow cytometers Aria III, Astrios EQ, and Canto II demonstrated superior performance in distinguishing ASC speck+ cells from diffuse ASC cells.
• Imaging flow cytometry and HCI provided valuable image-based parameters for assessing ASC speck formation.

Conclusions:

• Various technical methods can detect inflammasome activation via ASC speck formation, but results are device-dependent.
• The ability to differentiate diffuse ASC from ASC speck+ cells is critical for accurate inflammasome detection.
• High-content imaging (HCI) offers advantages like high-throughput and algorithm-driven quantification for ASC speck analysis.