Figure 2.

Live bacteria is critical for triggering bactericidal responses. (A) Phagocytosis of FITC-Ec or FITC-HK-Ec was determined after 60 min by flow cytometry. Dot plots showing the percentage and a histogram showing the MFI of FITC+ PMN of a representative experiment out of three are depicted. (B) ROS generation. Isolated PMN were stained with DHR for 15 min and then treated with Ec, HK-Ec, or I-Ec in a PMN:bacteria ratio of 1:20 for 30 min. The % of DHR+ PMN was determined by flow cytometry. Results were expressed as the mean ± ES, n = 8. Right panel: Representative dot-plots of the percentage of DHR+ PMN are shown in non-treated (−), Ec and HK-Ec-treated PMN. (C) Isolated PMN were incubated with Ec, HK-Ec, or I-Ec in a PMN:bacteria ratio of 1:20 for 3 h and NETs were stained with propidium iodide for DNA and with a specific antibody for elastase detection and were visualized by confocal microscopy. NETs area (μm²) was measured using the FIJI software as described in material and methods. Results were expressed as the mean ± ES, n = 10. Inset: Elastase activity using a specific substrate was determined and the absorbance (A) at 450–550 nm is depicted for untreated PMN (−) and PMN treated with Ec or HK-Ec. Results were expressed as the mean ± ES, n = 10. Right panel: Representative microphotographs of confocal images showing PMN from the untreated (−), Ec and HK-Ec groups stained for DNA or elastase visualization and the merge of the images (x200). In all cases ^*p < 0.05 vs. all other groups.