A novel real-time imaging technique to quantify mechanisms of cell death in neutrophils

Abstract

Abstract Neutrophils have distinct mechanisms of cell death, including the formation of neutrophil extracellular traps (NETs- a process called NETosis). NETosis is characterized by reactive oxygen species generation, chromatin decondensation, membrane rupture and extrusion of a meshwork of chromatin bound to granule peptides. Objective, efficient techniques to quantify NETosis are required. We developed a new method to automatically quantify the percentage of neutrophils undergoing NETosis using the IncuCyte ZOOM™ (Essen BioScience, Inc.) platform to image neutrophils at 20× in a 96-well plate and a dual-dye system dependent on membrane integrity to stain DNA, collecting both phase and fluorescence images. DNA in healthy control neutrophils were stained with a cell membrane permeable NUCLEAR-ID Red DNA dye. Neutrophils were then incubated with Sytox, a membrane-impermeable DNA dye, used to assess cell death. Conditions were run in triplicate and 3 images per well were captured at 10-minute intervals. A processing definition was set and optimized to count all neutrophils (NUCLEAR-ID stained) and neutrophils undergoing cell death (Sytox stained) using fluorescence intensity and stained area size to identify cells undergoing NETosis. Changes in nuclear morphology, quantified by the processing definition, distinguished PMA, Ionomycin and SLE sera induced NETosis from staurosporine induced apoptosis. Findings were confirmed and correlated with the gold-standard of immunofluorescence microscopy. This novel real time assay distinguishes types of neutrophil cell death and quantifies NETosis in a rapid, accurate and reproducible way, significantly improving the study of neutrophils.