Rhodamine phalloidin

Bacteria were inoculated with an MOI of 100 bacteria per host cell into cultured HeLa cells on glass coverslips in a 6-well plate and incubated at 37 °C for 4 h, then the medium was replaced by a fresh medium. The culture was continued for another 2 h. The coverslips were washed with PBS+. Rhodamine phalloidin (Life Technologies, Carlsbad, CA, United States) was used to visualize actin, and Hoechst33342 (Dojindo Laboratories, Tokyo, Japan) was used to stain bacteria and HeLa nuclei. Fluorescent images were acquired in the DAPI and Rhodamine phalloidin laser units on an Olympus FV1200 IX81 microscope using a 100 × objective and captured with a CCD camera.

Cells were allowed to attach for 8 h to glass coverslips. They were then fixed in 3.7% formaldehyde in PBS, permeabilized with 0.5% Triton X-100 and blocked with 1% BSA (Fraction V) in PBS for 1 h. Coverslips were incubated with the indicated antibodies at a 1∶500 dilution, or with rhodamine-phalloidin (Molecular Probes, Eugene, OR, USA) at a 1∶40 dilution in PBS for 20 min. Antibody-treated cells were washed in PBS and incubated with FITC or Texas red-conjugated secondary antibodies (Pierce Biotechnology, Rockford, IL, USA) at a 1∶500 dilution in PBS for 1 h. Cell nuclei were stained with DAPI (Vector Labs, Burlingame, CA). Finally, the cells were mounted using glycerol. Cells probed with rhodamine-phalloidin were washed and immediately mounted and observed by FV1000 laser scanning confocal microscopy (Olympus, Tokyo, Japan).