Tms f

Streptococcal strains were grown to the exponential phase and centrifuged at 5000×g for 5 min. Pelleted cells were then resuspended in 10% FBS α-MEM containing no antibiotics. Epithelial cells (2×10⁵ cells) in 24-well culture plates (Asahi Glass, Tokyo, Japan) were infected with viable streptococcal strains at a multiplicity of infection (MOI) of 50, 100, or 200, in the absence of antibiotics, for 2 h. Cells were washed with phosphate buffered saline (PBS, pH 7.2) to remove extracellular non-adherent bacteria, and cultured for 18 h in fresh medium containing antibiotics. Cells were then stained with 0.2% trypan blue (Sigma Aldrich, St. Louis, MO, USA) in PBS, and the numbers of viable and dead cells were counted using light microscopy (Nikon TMS-F, Nikon, Tokyo, Japan). One additional measure of cell death was whether the cells detached from the culture plates. The morphological changes of the infected cells were also determined using a phase-contrast microscope (Axiovert 40C, Carl Zeiss, Oberkochen, Germany). Cell death induced by H₂O₂ was determined using similar methods. Epithelial cells were treated with 1, 5, or 10 mM H₂O₂ (Nacalai Tesque, Kyoto, Japan) for 2 h, washed with PBS, and cultured for 18 h in fresh medium. The viability was determined by trypan blue staining.

The RBL-2H3 cells (2 × 10⁵ cells in 5% FBS DMEM) were infected with the viable streptococcal strains at an MOI of 200, in the absence of antibiotics, for 3 h. The culture medium was changed to a fresh medium containing antibiotics, and cultured for an additional 18 h. The cells were then stained with 0.2% trypan blue in PBS. After incubation at room temperature for 10 min, the numbers of viable and dead cells were counted using a microscope (Nikon TMS-F; Nikon, Tokyo, Japan). Because the dead RBL-2H3 cells were easily detached from the bottom of the culture plates, cells that disappeared during the washing and staining steps were considered to be dead (see S1 Fig). Cell death induced by H₂O₂ (2 mM) or PMA (10 nM) + ionomycin (1 μM) was determined similarly. To evaluate the dose-dependent effect, the cells were infected with viable S. oralis WT (MOI = 10, 50 or 200), or treated with H₂O₂ (0.1, 0.5 or 2 mM).
The effect of catalase was also investigated. Prior to infection, 10, 50 or 200 U/mL of catalase was added to the culture of RBL-2H3 cells, and the cells were then infected with viable S. oralis WT (MOI = 200) for 3 h. The cells were washed with PBS and cultured in fresh medium containing catalase and antibiotics for 18 h. The viability was determined as described above.

This method was performed as described previously^{17 (link)}. Cells (1 × 10⁶ cells per well) were plated on 6-well plates. Cells were cultured in medium containing 1% bovine serum albumin (BSA). A sterile 200 μl pipette tip was used to create a gap by scraping the cells. The migration process was monitored (after identification of each wounded zone) in six areas immediately and 48 h after wounds were made using an inverted microscope (Nikon TMS-F, 301655, Nikon, Tokyo, Japan) installed with a digital camera (Nikon Digital shot DS-L1, Nikon). Cell migration data were expressed as the ratio of the change in gap width divided by the initial gap width.