Quantification of Neutrophil Extracellular Traps

NETs were enumerated using three different techniques: 1. a SYTO/SYTOX staining technique. The cell impermeable SYTOX orange dye (1 μM) was used to detect NETs. SYTO green, a cell-permeable DNA dye (250 nM), was used to determine the total number of cells. Images were taken on a Leica DM IRBE inverted microscope. 2. Immunofluorescence staining as described above, followed by automated quantification of NETs using ImageJ. The Hoechst signal was used to calculate the total amount of cells per microscopic field and NETs were quantified by the PL2-3 signal (chromatin) accounted for nuclear expansion [10 (link)]. 3. By using the cell impermeable DNA dye SYTOX green (50 nM) and measuring in a fluorometer with an excitation/ emission of 485/518 nm, respectively.