Porphyromonas gingivalis

Gingival fibroblasts, established from gingival biopsies obtained from three individuals with no clinical signs of periodontitis, were cultured as described previously67 (link). Human primary gingival epithelial cells were purchased from CellnTec (CellnTec Advanced Cell Systems AG, Bern, Switzerland) and cultured in CnT-PR cell culture medium (CellnTec) at 37 °C in a 5% CO₂ incubator. The cells (0.5 × 10⁶) were seeded in Petri dishes (60 mm) in DMEM supplemented with penicillin (50 units/ml), streptomycin (50 μg/ml), and 5% fetal calf serum (FCS, Technologies Europe BV) in case of the fibroblasts, and in iCnT-PR cell culture medium (CellnTec) in case of the epithelial cells, and cultured at 37 °C for 24 hours. The cell layers were rinsed twice with serum-free medium (DMEM or CnT-PR medium) followed by the addition of 2.0 ml of medium in the absence or presence of 5 μg/ml Porphyromonas gingivalis or E.coli LPS (InvivoGen, Toulouse, France). After incubation for 24 hours, the culture medium was collected and the cells washed twice with ice-cold PBS and used for total isolation of RNA.