Imaging Internalization of Bacteria in Macrophages

The bacterial suspension (at a concentration of 10⁹ CFU/mL) was incubated with SytoRed (20 uM, dilution 1:250) (Molecular Probes, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature. After three washes with PBS, the stained bacteria were resuspended in 1 mL of RPMI without FBS and antibiotics. THP-1 cells were seeded in 96-well plates and differentiated for 48 h with PMA. Then, THP-1-derived macrophages were incubated with different concentrations of TB (10⁶, 5 × 10⁶, 10⁷, 5 × 10⁷, 10⁸ CFU/mL) for 30 min. At the end of the incubation, the cells were washed with PBS, fixed in 4% paraformaldehyde for 5 min and washed again with PBS. After that, the cells were stained with Actin Green (Molecular Probes, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), which binds actin with high affinity. Nuclei were stained with Hoechst (Molecular Probes, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), diluted 1:300 in PBS, as previously described [51 (link)], and analyzed by the Operetta CLS system (PerkinElmer, Waltham, MA, USA) in the confocal mode at 63× magnification.