Video-Fluorescence Microscopy

Neutrophils were isolated as described in section Isolation of bovine PMNs. Again, 19,500 cells were seeded in a 12-well silicone chamber slide (Ibidi) and preincubated for 1 h at 37°C and 5% CO₂. To detect DNA, neutrophils were incubated at 37°C for 20 min with Hoechst 33342 (final concentration 10 μg/mL, Sigma Aldrich) prior to a washing step with RPMI 1640. The medium was then exchanged once more and mucins (20 μg/μL) were added to the cells. Subsequently, cells were analyzed for 4 h using fluorescence microscopy (Carl Zeiss confocal laser scanning microscope LSM 800). Cells were monitored using a shooting interval of 1 picture/min.