Tlr9 ligand type b cpg

RASF and OASF were cultured as previously described [5] (link), and were used at passages 4–6. For TLR stimulation, 5×10⁴ RASF per well in 6-well plates were incubated with different TLR ligands for 4 h and 24 h, respectively, as follows: TLR2 ligand peptidoglycan (PGN, 10 µg/ml), TLR3 ligand polyinosinic:polycytidylic acid (ploy(I:C), 25 µg/ml), TLR4 ligand lipopolysaccharide (LPS, 100 ng/mL) from Sigma; TLR9 ligand Type B CpG (10 µg/ml) from Invitrogen (Carlsbad, CA, USA). The cells were harvested for RT-PCR and realtime PCR while the supernatants were collected for ELISA.

Synovial tissue samples were minced mechanically, washed with phosphate-buffered saline and seeded into tissue culture flasks for 2 h at 37 °C until the fragments attached to the plates. Then, Dulbecco’s modified Eagle medium (HyClone, Logan, UT) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Carlsbad, CA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA) was added to the tissue culture flasks and the cells cultured in a humidified incubator containing 5% CO₂ at 37 °C. After overnight culture, nonadherent cells were removed and adherent cells were cultured. Cells from passages 4 to 7 were used. The purity of synovial fibroblast cells was more than 95% according to CD90 expression assessed using flow cytometry. For TLR ligand stimulation, RASF (5 × 10⁴/well) in 6-well plates were incubated with different TLR ligands for 24 h, respectively, as follows: TLR2 ligand peptidoglycan (PGN; 10 μg/mL), TLR3 ligand polyinosinic:polycytidylic acid (ployI:C; 25 μg/mL), TLR4 ligand lipopolysaccharide (LPS; 100 ng/mL) from Sigma (St. Louis, Missouri), and TLR9 ligand Type B CpG (10 μg/mL) from Invitrogen (Carlsbad, CA, USA). The cells were harvested for the next experiment.