Flow cytometric and multimodal detection of ASC speck formation in THP-1 monocytes and murine macrophages following canonical inflammasome activation

The detection of inflammasome activation plays a critical role in understanding innate immune responses across various diseases. Apoptosis-associated speck-like protein containing a CARD (ASC) speck formation serves as a direct indicator of inflammasome assembly; however, standardized detection protocols remain scarce. In this study, we evaluated ASC speck formation in THP-1 human monocytes using flow cytometry, fluorescence microscopy, and immunoblotting under canonical NLRP3 inflammasome stimulation conditions. THP-1 cells were first primed with lipopolysaccharide (LPS) and then stimulated with nigericin (Nig), which resulted in a significant increase in ASC speck-positive monocytes (from 4.86% to 15.03%, p < 0.01). Fluorescence microscopy confirmed punctate ASC localization, and Western blot analysis revealed ASC oligomer formation. These findings demonstrate that a flow cytometry-based ASC speck assay offers a sensitive and practical method for assessing inflammasome activation, establishing a robust platform for translational immunology research.