Raw264.7 cells

Line-1 cells from the BALB/cByJ alveolar lung carcinoma (provided by Dr. John Yuhas) were adapted to tissue culture [48 (link)]. Line-1 cells were cultured in RPMI Medium 1640 (Gibco, New York, USA) supplemented with 10% fetal bovine serum (Gibco). HepG2 cells (human hepatocellular carcinoma cell line) and RAW264.7 cells (Murine macrophage cell line) were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). HepG2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco), supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino acids (NEAA) (Gibco), and 1% streptomycin-penicillin (100 IU/mL) (Hyclonr, USA). RAW264.7 cells were grown in DMEM supplemented with 10% fetal bovine serum. All cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ for varying times.

RAW264.7 cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (HyClone, Logan, UT), 2 mM glutamine, 1% nonessential amino acid, 1 mM pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen Life Technologies, Carlsbad, CA). Cells were maintained in a humidified incubator at 37°C in 5% CO₂.

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).

RAW264.7 cells was obtained from the Bioresource Collection and Research Center and maintained in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% heat-inactivated bovine calf serum (Sigma-Aldrich, SIAL, USA) with 100 units/ml penicillin, 100 μg/mL streptomycin, and 0.3 mg/mL of L-glutamine (PSG, Thermo Fisher Scientific) and 1 mM sodium pyruvate (Thermo Fisher Scientific) at 37 °C in 5% CO₂ incubator. BMDMs were cultured in RPMI medium 1640 (Thermo Fisher Scientific) supplemented with PSG, non-essential amino acids (NEAA, Thermo Fisher Scientific), 10% fetal bovine serum (Thermo Fisher Scientific), and 20 ng/mL M-CSF (R&D Systems, MN, USA). CD4⁺ T cells and stimulated BMDMs were cultured in RPMI medium 1640 supplemented with PSG, NEAA, 10% fetal bovine serum, and mIL-2 (20 ng/mL, Peprotech, Rocky Hill, USA). For detection of IL-2, mIL-2 was removed from the complete medium.

Raw 264.7 cells were purchased as cryopreserved cultures from Bioresource Collection and Research Center (Hsinchu, Taiwan). 1 × 10⁶ cells were seeded into 100 mm Petri dishes and incubated at 37°C in 90% Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose + 10% fetal bovine serum. Primary human bladder smooth muscle cells (HBdSMC) (ScienCell, CA, USA) were grown in the smooth muscle cell medium (SMCM, Catalog number 1101). By which time the cells had reached 90% confluence, the cells were plated at 96-well or 10 cm plates for MTT assay or Western blot [14 (link), 15 ], respectively.

RAW264.7 cells (Bioresource Collection and Research Center, Hsin-Chu, Taiwan) were cultured with a medium (high-glucose DMEM (Gibco, Grand Island, NY, USA), 10% FBS, and 1% P.S.) in an atmosphere containing 5% CO₂ at 37°C. RAW264.7 cells were pretreated with various concentrations of GSYJ in the presence or absence of 100 ng/ml LPS. The cells were harvested at the indicated time points for quantitative PCR and western blotting. Cell supernatants were collected for ELISA and NO measurements.