Bcrc14417

The P. gingivalis strain BCRC14417 was obtained from the Bioresource Collection and Research Center, Hsinchu, Taiwan. Bacterial suspensions to induce periodontitis of mice were prepared by a method described elsewhere [13 (link)]. Briefly, P. gingivalis was cultured anaerobically in tryptic soy broth (TSB, Difco, Detroit, MI, USA) supplemented with 2.5% yeast extract, hemin, and menadione at 37 °C. The numbers of bacteria were determined with a spectrophotometer (at an optical density at 600 nm) based on a standard curve established by colony formation on bacterial plates. To prepare heat-inactivated P. gingivalis, bacterial suspensions in phosphate-buffered saline (PBS) were heated at 80 °C for 30 min, washed with PBS, and re-suspended in RPMI 1640 medium (Gibco, Carlsbad, CA, USA).

The strain of Porphyromonas gingivalis (BCRC14417) was obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Fresh P. gingivalis cells from blood agar plates supplemented with hemin (5 μg/mL; Sigma-Aldrich, St Louis, MO, USA) and menadione (1 μg/mL; Sigma-Aldrich) were inoculated into 5 mL of trypticase soy broth (TSB, Difco, Detroit, MI, USA) supplemented with 2.5% yeast extract, hemin, and menadione. The cultures were incubated anaerobically in an N₂-H₂-CO₂ (80:10:10) atmosphere at 37 °C by using a Concept 400 Anaerobic Workstations (Ruskinn, Sanford, ME, USA). Organisms were harvested by centrifugation, washed with phosphate-buffered saline (PBS), and re-suspended in PBS. The numbers of bacteria were determined with a spectrophotometer (at an optical density at 600 nm) based on a standard curve established by colony formation on bacterial plates. To prepare heat-killed P. gingivalis cells, bacterial suspensions in PBS were heated at 80 °C for 30 min, washed with PBS, and re-suspended in RPMI 1640 medium (Gibco, Carlsbad, CA, USA).

P. gingivalis BCRC 14417, A. actinomycetemcomitans BCRC 80375, and RAW 264.7 cells were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), l-glutamine, LPS, and receptor activator of nuclear factor-κB ligand (RANKL) were all purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Brain heart infusion agar, Mueller-Hinton agar, and tryptic soy broth were purchased from BD Co. (Franklin Lakes, NJ, USA). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from HyClone Laboratories (Logan, UT, USA). A tartrate-resistant acid phosphatase (TRAP) & alkaline phosphatase (ALP) Double-stain Kit were purchased from Takara Bio Inc. (Kusatsu, Shiga, Japan) and pro-inflammatory cytokine assay kits (IL-1β, IL-6, IL-17, and TNF-α) were purchased from BioLegend (San Diego, CA, USA).