Background Neutrophils play a key role in the pathogenesis of autoimmune diseases through various mechanisms including the formation of neutrophil extracellular traps (NETs). NETosis, a recently described distinct form of program neutrophil cell death, is characterized by reactive oxygen species generation, chromatin and nuclear decondensation, membrane rupture and extrusion of a meshwork of chromatin bound to granule peptides.
Objectives Techniques to assess and quantitate NETosis in an unbiased, reproducible and efficient way are lacking. We developed a new method to automatically quantify the percentage of neutrophils undergoing NETosis using real-time quantitative live-cell analysis with IncuCyte ZOOM™ (Essen BioScience, Inc.) platform and a dual-dye system dependent on membrane integrity to stain DNA, to image neutrophils and characterize their mechanisms of cell death.
Methods Neutrophils were isolated from healthy controls using density gradient methods and their DNA was stained with a membrane permeable NUCLEAR-ID Red DNA dye. Neutrophils were plated and incubated with various stimuli to induce NETosis (PMA, ionomycin and/or SLE sera), apoptosis (Staurosporin) or necroptosis (TNF with a pan-caspase inhibitor, Z-VAD) and with Sytox, a membrane-impermeable DNA dye. Three 20x magnification images from different areas per well were captured at 10-minute intervals. A processing definition was set and optimized to count all neutrophils (NUCLEAR-ID stained) at baseline and neutrophils undergoing cell death (Sytox stained) at three hours using fluorescence intensity and stained area size (Figure 1).
Results Percentage of neutrophils undergoing cell death using various stimuli was highly reproducible. Characterization of changes in nuclear morphology, quantified by the processing definition, distinguished between NETosis, apoptosis and necroptosis. Findings were confirmed and counts correlated with previously established method using immunofluorescence microscopy.
Conclusions This novel real time assay distinguishes types of neutrophil cell death and quantifies NETosis in a rapid, accurate and reproducible way. This technique may facilitate studies in neutrophil biology.
Disclosure of Interest None declared