Nico Wittmann, Sander Bekeschus, Doreen Biedenweg, Daniela Kuthning, Christopher Pohl, Jana Gramenz, Oliver Otto, Lukas Bossaller, Almut Meyer-Bahlburg

• Inflammasome activation occurs in various diseases, including rare diseases that require multicenter studies for investigation. Flow cytometric analysis of ASC speck + cells in patient samples can be used to detect cell type-specific inflammasome activation. However, this requires standardized sample processing and the ability to compare data from different flow cytometers. To address this issue, we analyzed stimulated and unstimulated PBMCs from healthy donors using seven different flow cytometers. Additionally, human PBMCs were analyzed by fluorescence microscopy, imaging flow cytometry and high-content imaging (HCI). Flow cytometers differed significantly in their ability to detect ASC speck + cells. Aria III, Astrios EQ, and Canto II performed best in separating ASC speck + from diffuse ASC cells. Imaging flow cytometry and HCI provided additional insight into ASC speck formation based on image-based parameters. For optimal results, the ability to separate cells with diffuse ASC from ASC speck + cells is decisive. Image-based parameters can also differentiate cells with diffuse ASC from ASC speck + cells. For the first time, we analyzed ASC speck detection by HCI in PBMCs and demonstrated advantages of this technique, such as high-throughput, algorithm-driven image quantification and 3D-rendering. Thus, inflammasome activation by ASC speck formation can be detected by various technical methods. However, the results may vary depending on the device used.