Xcelligence rtca dp instrument

The RTCA instrument (xCELLigence RTCA DP instrument; Roche Diagnostics GmbH, Mannheim, Germany) was used to analyze the proliferation properties of gingival fibroblasts and osteoblasts with the different experimental approaches. Based on preliminary results, 10 × 10³ osteoblasts and 5 × 10³ fibroblasts were used. Initially, 100 µl of cell-free growth medium was added to the wells of a 16x microtiter plate (E-Plate, Roche Diagnostics GmbH, Mannheim, Germany) using different supplements. After 30 min, the background impedance was measured for each well. Subsequently, 50 µl of the cell suspension with the desired number of cells was added to each well. According to the manufacturer’s guidelines, the plate was left at RT for 30 min to allow cell adhesion before being locked into the RTCA DP device incubator. The adhesion, spread and proliferation of the cells were monitored in 15 min intervals. The experiment was carried out for 72 h. The impedance of the cell sensor was described and measured as the cell index (CI). The CI value at each time point is defined as Rn–Rb/Rb, where Rn is the cell electrode impedance of the well and Rb is the background impedance of the well alone with the medium.

The xCELLigence RTCA DP instrument (Roche) was used for the real-time monitoring of cell growth. Cells were seeded at 20,000 cells per well in E-Plate 16 plates (ACEA Biosciences, UK). Impedance measurements for each well were then taken automatically every 10 min and expressed as a CI.

ST cells were seeded on a 16-well E-Plate with 5000 cells per well and allowed to grow for 12–24 h. The cells were transfected with miR-762 mimic or miR-762 inhibitor when the cell index reached 1.0–2.0 with three wells per treatment. Cell growth and proliferation were monitored by an xCELLigence RTCA DP instrument (Roche Applied Science, Penzberg, Upper Bavaria, Germany).
The cell cycle was analysed with the Cell Cycle Detection Kit (KeyGEN BioTECH, Nanjing, China). Briefly, 48 h after transfection, the ST cells were fixed in 70% (v/v) ethanol overnight at −20 °C. Following incubation in 50 mg/ml propidium iodide (PI) for 30 min at 4 °C, the cells were analysed using FACSCalibur Flow Cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) and the ModFit software (Verity Software House). The proliferative index was derived by estimating the proportion of mitotic cells from a total of 20,000 cells.