Zen microscopic software

PMA-differentiated THP-1 cells were preincubated with 2 mg/mL of E. faecalis for 24 h or with 10 μM cytochalasin D for 1 h, followed stimulation with YG beads (10 beads/cell), fecal content, E. coli (MOI 100) or P. mirabilis (MOI 100) for 3 h. Cells were stained for visualization of tubulin (1:100 dilution of anti-α-tubulin followed by a 1:1000 dilution of Alexa Fluor-594 conjugated secondsondary antibody (Invitrogen, red), YG beads, fecal content, E. coli or P. mirabilis (CFSE, green) and nuclei (DAPI, blue). To demonstrate the phagocytic efficiency of THP-1 cells, images were obtained with a confocal laser scanning microscope (LSM780; Carl Zeiss, Jena, Germany) and processed with the ZEN microscopic software (Carl Zeiss). The phagocytic activity was quantified by calculating the percentage of cells with internalized YG beads, CFSE-labeled fecal content or bacteria, as assessed using an IN cell analyzer (GE Healthcare, Freiburg, Germany).

Cells were treated with MitoTracker Green FM (200 nM) and Hoechst (5 μg/ml) for 30 min. To measure the cellular distribution of mitochondria, images were obtained with a confocal laser scanning microscope (Carl Zeiss, LSM780) and processed with the ZEN microscopic software (Carl Zeiss).