Fig. 2.

PCa induces neutrophil oxidative burst and NET release. a Neutrophils incubated for 3 h in conditioned media (CM) from RWPE, LNCaP and C42B cell lines supplemented with 5 nM dihydrorhodamine 123 (DHR123) and DAPI. Scatter plots show oxidative burst as expression of green fluorescence (x-axis) compared to DAPI (measure of viability) (y-axis) of CM-treated neutrophils compared to serum-free base media (negative control) and complete media, supplemented with 10% FBS and 5 μg/ml LPS (positive control). Images shown are representative from 1 of 4 repeat experiments. b Mouse bone marrow neutrophils incubated in CM for 2 h (n = 3 wells/media) and Sytox Green, DNA-binding dye for an additional 20 min. Images (left) were taken at a 10x objective under a GFP laser excitation using the EVOS Auto Light Microscope. White arrows denote “string-like” DNA-containing NETs. Quantitation of NETs (graph) were defined as the percentage of NETs per total cell number per well. Asterisks denote statistical significance (**p < 0.01, ***p < 0.001, p < 0.0001)