, 2003; Giacona et al., 2004). Approximately 100 cells per well were examined using a microscope (×200) (Nikon DIAPHOT TMD300; Nikon, Tokyo, Japan). Differentiated THP-1 macrophages were infected with viable S. sanguinis SK36 (MOI; 50, 100
or 200) or S. mutans UA159 in the absence of antibiotics. After 2 h of incubation, the cells were washed three times with PBS, and were disrupted by vortexing MS-275 with sterile water. Serial dilutions of the cell lysates were plated onto BHI agar plates to determine the number of adherent bacteria (CFU). For the internalization assay, the extracellular adherent bacteria were killed by incubating with gentamicin (100 μg mL−1) and penicillin G (100 U mL−1) for 1 h. The cells were then lysed with sterile water and CFU of intracellular bacteria were counted on BHI agar plates (Okahashi et al., 2003). Differentiated THP-1 macrophages (2 × 105 cells in 5% FBS RPMI1640) were infected with viable S. sanguinis SK36
(MOI 50, 100 or 200) or heat-inactivated S. sanguinis (MOI 500 or 1000) in the absence of antibiotics for 2 h. The cells were washed with PBS and cultured for 18 h in fresh medium containing antibiotics. The cells were then stained with 0.2% trypan blue (Sigma Aldrich) in PBS. After incubation at room temperature for 5 min, the numbers of viable and dead cells were counted using a microscope (Nikon TMS-F; Nikon). Differentiated THP-1 cells were cultured on gelatin-coated Smoothened inhibitor coverslips in 24-well culture plates. The macrophages were exposed to S. sanguinis SK36 at an MOI of 200 for 2 h, washed with PBS to remove extracellular during bacteria, and cultured for a further 6 h. Prolonged incubation resulted in detachment of the dead macrophages from the coverslips. Uninfected cells were used as a negative control. The cells were first stained with propidium iodide (PI) (Sigma Aldrich), washed with PBS, treated with 0.1% Triton X100 in PBS for 10 min, and then stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma Aldrich). The stained cells were
analyzed using an LSM 510 confocal laser microscope (Carl Zeiss, Oberkochen, Germany). PI stained the nuclear DNA of dead THP-1 cells, whereas DAPI stained that in all cells. Differentiated THP-1 macrophages were infected with viable S. sanguinis SK36 (MOI 50, 100 or 200) or heat-inactivated S. sanguinis (MOI 500 or 1000) in the absence of antibiotics for 2 h. The cells were washed with PBS to remove extracellular bacteria, and cultured in fresh medium containing antibiotics for a further 18 h. As a stimulant, E. coli LPS (1 μg mL−1) was also utilized. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the culture supernatants were measured using enzyme-linked immunosorbent assay kits (ELISA; Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Culture supernatants of differentiated THP-1 macrophages were prepared as described above.