Fig. 2.

Overview of fluorescence images of NETs with subsequent evaluation by hand counting or semi-automated quantification.

NET-negative cells are morphologically seen as small, round, or lobulated nuclei that are stained with DAPI (a blue-fluorescent DNA stain). NET-positive cells show green extracellular off-shoots including any cells that attach to the NET or are distinguished by their blurry rim, enlarged and decondensed (puffy) green nucleus. Green indicates positive staining for DNA-histone-1-complexes as marker for NETs. For hand counting, all cells were counted with the Cell Counter plugin for ImageJ for individually marking each cell. Here, NET-negative cells were labeled in magenta with the number 4, and NET-positive cells were marked in yellow with the number 5. The workflow of NET quantification with the semi-automated approach is shown in ImageJ. Images were separated into respective channels for DAPI (blue) and DNA/Histone complex (green). After converting to the 8-Bit format, values for the background threshold and minimal size were determined. The program determined the events for each channel and respective values were used for manual calculation of NET formation rates.