Microscope cover glass

ZF4 cells and Lm EGD-e △actA/inlB (pERL3-29) were co-cultured in a microscope cover glass (NEST, China) at a cell carbon dioxide incubator under 28 °C for 1 h. ZF4 cells without treatment were regarded as a control group. A volume of 0.5 mL of the gentamicin (200 µg/mL) was added to cells at 28 °C for 30 min to kill extracellular bacteria. Subsequently, the mixture was washed three times with PBS, and the cells were fixed with 0.5 mL of 4 % paraformaldehyde in PBS at room temperature for 30 min. After the mixture was washed three times with PBS, the cells were permeabilized with 0.1 % Triton X-100 in PBS for 5 min. Then, the mixture was washed three times with PBS, and the actin of the cells was stained with iFluor™ 555 phalloidin (YEASEN, Shanghai, China) at room temperature and kept in dark the place for 90 min. After the mixture was washed three times with PBS, cell nucleus were stained with DAPI (YEASEN, Shanghai, China) at room temperature and kept in the dark place for 5 min. Finally, the mixture was washed twice with PBS, and images were observed and captured on Leica DM 2500 fluorescence microscope (Leica, Germany).