Simple SummaryThe involvement of the innate immune system in autoimmune diseases, such as type 1 diabetes, is becoming more and more apparent. Perhaps the most overlooked innate immune cell type, the neutrophil, is one of the culprits for hyperactive immune responses (against oneself) in these diseases. However, the heterogeneous nature, both within and between these diseases, and the lack of standardized methods to study neutrophil function has hampered advancement in this research area. Our study used a live-cell imaging technique that allows for an unbiased and automated analysis of neutrophil function in children and adults with type 1 diabetes. Overall, neutrophils of people at different developmental stages of type 1 diabetes, irrespective of age, behaved similarly to those of healthy donors, despite some minor changes in peripheral neutrophil measures.AbstractNeutrophils might play an important role in the pathogenesis of autoimmune diseases, including type 1 diabetes (T1D), by contributing to immune dysregulation via a highly inflammatory program called neutrophil extracellular trap (NET) formation or NETosis, involving the extrusion of chromatin entangled with anti-microbial proteins. However, numerous studies reported contradictory data on NET formation in T1D. This might in part be due to the inherent heterogeneity of the disease and the influence of the disease developmental stage on neutrophil behavior. Moreover, there is a lack of a standardized method to measure NETosis in an unbiased and robust manner. In this study, we employed the Incucyte®ZOOM live-cell imaging platform to study NETosis levels in various subtypes of adult and pediatric T1D donors compared to healthy controls (HC) at baseline and in response to phorbol–myristate acetate (PMA) and ionomycin. Firstly, we determined that the technique allows for an operator-independent and automated quantification of NET formation across multiple time points, which showed that PMA and ionomycin induced NETosis with distinct kinetic characteristics, confirmed by high-resolution microscopy. NETosis levels also showed a clear dose-response curve to increasing concentrations of both stimuli. Overall, using Incucyte®ZOOM, no aberrant NET formation was observed over time in the different subtypes of T1D populations, irrespective of age, compared to HC. These data were corroborated by the levels of peripheral NET markers in all study participants. The current study showed that live-cell imaging allows for a robust and unbiased analysis and quantification of NET formation in real-time. Peripheral neutrophil measures should be complemented with dynamic quantification of NETing neutrophils to make robust conclusions on NET formation in health and disease.