Quantification of Neutrophil Extracellular Traps

NET formation was also quantified by detecting DNA release spectrophotometrically with the DNA-binding dye SYTOX® Green as previously described (Behnen et al., 2014 (link); Gonzalez et al., 2014 (link); Alemán et al., 2016a (link),b (link)). Briefly, neutrophils were resuspended at 1 × 10⁶ cell/ml in RPMI-1640 medium (Gibco®; Grand Island, NY), containing 500 nM SYTOX® Green (Molecular Probes, Inc.; Eugene, OR). Then, 100 μl of this cell suspension (1 × 10⁵ PMN) were added to each well of a 96-well plate (Costar® 3590; Corning Inc., Corning, NY). Next, the plate was incubated at 37°C in a 5% CO₂ incubator for 20 min. Neutrophils were then stimulated by adding 20 μl of 120 nM PMA (20 nM final concentration), or 20 μl of an E. histolytica suspension (2.5 × 10⁵ amoeba/ml) to each corresponding well. The amoeba to neutrophil ratio was 1:20. The plate was then incubated in a 35°C pre-warmed microplate reader, model Synergy HT from BioTek Instruments (Winooski, VT), for up to 4 h. For this assay, cells were not fixed. The fluorescence from the bottom of the plate was read every 5 min, using the 485 nm excitation and 528 emission filters.