Neutrophils are the most abundant immune cells in the blood. Besides common immune defense mechanisms, releasing their DNA covered with antimicrobial proteases and histones represent another strong defense mechanism: neutrophil extracellular traps. In vitro the two most common inducers of these, so called, NETs are calcium ionophores (CI) and PMA (Phorbol 12-myristate 13-acetate). Following stimulation monitoring of NET release is necessary. For now, the methods of choice are quantification of free DNA by fluorescent dyes or analysis of immunofluorescence images. As a new method we tested bio-impedance monitoring of neutrophils after stimulation with the two inducers PMA and CI in gold-electrode coated plates. Bio-impedance (cell index) was measured over time. Results were compared to the monitoring of NETs by the fluorescent DNA-binding dye Sytox Green and immunofluorescence analysis. Cell index peaked about 25 min faster following CI stimulation than following PMA stimulation. The activation in Sytox Green Assay was significantly later detectable for PMA (+ approx. 90 min) but not for CI stimulation. The earlier and faster activation by CI was also confirmed by immunofluorescence staining. Our data suggest that bio-impedance measurement allows an easy online tracking of early neutrophil activation. This offers new opportunities to monitor early phases and stimuli-dependent dynamics of NETosis.
Keywords: Sytox Green Assay; bio-impedance; immune cell activation; neutrophil extracellular traps; neutrophils; xCelligence.
Copyright © 2020 Linnemann et al.