Fluorescence and immunofluorescence microscopy of stimulated human neutrophils for NETs formation. Stimulated normal human neutrophils were seeded on poly-l-lysine–coated slides and incubated at 37°C + 5% CO₂ for 3 hours. After incubation, the preparations were fixed using 4% paraformaldehyde and stained using 2.5 μM Sytox Green, a DNA staining dye. Unstimulated neutrophils did not form NETs (A) and were intact after treatment with DNAase (20 μM) (B). Robust NET release after stimulation with 600 nM PMA (C) or 5 μM A23187 (E) as shown by the bright and scattered Sytox Green signal pattern. Digestion of PMA-stimulated (D) and A23187-stimulated (F) NETs after treatment with DNAase (20 μg/mL), indicating the extracellular location of NETs. (G) Dual immunofluorescence staining of NETs by Sytox Green (2.5 μM) for DNA and citrillunated histone H3 (red). A23187, calcium ionophore.