This strain was grown at 37°C under anaerobic conditions on 5% horse blood agar plates (Poa Media, Eiken Chemical Co., Ltd., Tokyo, Japan) and in 30 mg/ml trypticase soy broth (BD Biosciences, SanJose, CA) supplemented with 2.5 mg/ml yeast extract (BD Biosciences), 5 μg/ml hemin and 5 μg/ml menadione. Bacterial growth was monitored by measuring the optical density at 660 nm (OD660). For invasion assays,
an inoculum with an infection ratio (multiplicity of infection [MOI]) of 100 bacteria per cell was added to the cell culture medium. Cell culture The human gingival this website epithelial cell line Ca9-22 was obtained from RIKEN Bioresource Center (Ibaraki, Japan). Ca9-22 cells were cultured under standard conditions in Eagle’s minimal essential medium (E-MEM; Wako Pure Chemical www.selleckchem.com/products/epoxomicin-bu-4061t.html Industries, Ltd., Osaka, Japan) containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin at 37°C in a humidified atmosphere
of 5% CO2. The monocytic cell line THP-1 was obtained from Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). THP-1 cells were cultured under standard conditions in Roswell Park Memorial Institute (RPMI) 1640 Medium (Invitrogen, Carlsbad, CA) containing 10% FBS, 1% penicillin and streptomycin at 37°C in a humidified atmosphere of 5% CO2. Antibodies Antibodies were obtained from Caspase Inhibitor VI the following sources: antiserum for P. gingivalis whole cells was kindly donated by Dr. Fuminobu Yoshimura (Aichi-gakuin University, Aichi, Japan); mouse monoclonal antibody specific for ICAM-1, goat polyclonal antibody specific for ICAM-1, mouse monoclonal antibody specific for TNFRI, mouse monoclonal antibody specific for TNFRII and mouse immunoglobulin G (IgG) (R & D Systems, Minneapolis, MN); mouse monoclonal antibody specific for Rab5 (BD Biosciences); rabbit polyclonal antibody specific for ICAM-1 (Santa Cruz Biotechnology, Dallas, TX); goat IgG (Alpha Diagnostic Intl. Inc.,
San Antonio, TX); mouse monoclonal antibody specific for β-actin (Biovision Exoribonuclease Inc., Milpitas, CA); anti-rabbit IgG-Alexa 555 and anti-rabbit IgG-Alexa 633 (Invitrogen); mouse monoclonal antibody specific for GFP (Novus Biologicals, Littleton, CO), anti-mouse IgG-HRP, anti-rabbit IgG-HRP and mouse monoclonal antibody specific for β-actin (Cell Signaling Technology, Danvers, MA). Vector constructs GFP-Rab5Q79L, GFP-Rab5WT, and GFP-Rab5S34N in pcDNA3 constructs were kindly provided by Dr. Yuji Yamamoto (Tokyo University of Agriculture, Tokyo, Japan) [57,58]. The GST-R5BD vector was kindly donated by Dr. Guangpu Li (University of Oklahoma Health Science Center, Oklahoma City, OK). P. gingivalis invasion assay Invasion of bacteria was quantitated by a standard antibiotic protection assay as described previously . Briefly, Ca9-22 cells were seeded in 12-well flat-bottom culture plates and were incubated overnight before administration of P. gingivalis.