Integrated real-time impedance and fluorescence imaging to characterize NETosis

NETosis is a specialized form of neutrophil cell death characterized by the release of web-like DNA structures and associated proteins, known as neutrophil extracellular traps (NETs). Two main pathways mediate NETosis¹ , ²: (1) classical “suicidal” NETosis, which involves cell lysis following NET release, typically occurs within 3-4 hours and depends on reactive oxygen species (ROS) generation via NADPH oxidase; and (2) “vital” NETosis, where NETs are extruded through vesicles within 1-2 hours while the cell remains intact. NETosis plays a dual role in host defense and disease, contributing to antimicrobial immunity as well as pathological inflammation, thrombosis, and autoimmunity³. In this study, differentiated neutrophil-like HL-60 (dHL-60) and primary human neutrophils were treated with a small set of compounds: PMA, which triggers suicidal NETosis; A23187, a pharmacological agent that induces rapid, vital NETosis; and camptothecin, an apoptotic compound. To visualize NET formation, the cells were cultured in media containing eTox Green, a membrane-impermeable DNA-binding dye. The morphological and physiological changes were continuously monitored via both impedance readout, reported as Cell Index, and live-cell imaging simultaneously on an xCELLigence RTCA eSight system. Our results show that: (1) the extent of DNA-binding dye staining distinguished apoptosis from NETosis, with NETosis inducers PMA and A23187 producing larger green fluorescence areas than the apoptotic agent camptothecin, reflecting that extruded NETs are substantially larger than nuclei; (2) suicidal NETosis induced by PMA was differentiated from vital NETosis triggered by A23187 based on ROS dependency-NET release was completely inhibited by DPI, an NADPH oxidase blocker, after PMA treatment but not after A23187 exposure. Additional distinctions included earlier onset of NET extrusion with A23187 and neutrophil death following PMA stimulation but not A23187 treatment; and (3) PMA-induced NETosis was associated with a transition from suspension to adherent states, which was monitored and quantified by impedance measurements. In conclusion, this real-time, noninvasive impedance-imaging approach enables visualization of NETosis, quantification of NET release kinetics via fluorescence imaging, and monitoring of neutrophil transitions between suspension and adherent states through impedance measurements. This integrated method provides a robust and versatile platform for mechanistic NETosis studies, drug-screening applications, and broader investigations into neutrophil-driven pathologies.