Xcelligence real time cell analysis system

The effect of KGN on hBMSCs proliferation was monitored using the xCELLigence Real-Time Cell Analysis (RTCA) system (Roche Applied Science, Penzberg, Germany). The procedure was carried out as previously described in our in vitro study [40 (link)]. Kartogenin (Sigma Aldrich, Saint Louis, MO, USA) used in this study was dissolved in dimethyl sulfoxide at a concentration of 5 mM. Different concentrations of KGN (0.1; 1; 10; 100 µM) were tested in order to determine the optimal concentration for our experiments. In brief, 3000 cells/well in 100 μL of culture medium (Alpha-MEM, 10% FBS, 1% ATB) in E-Plate 16 was exposed after 24 h to 100 μL of medium containing indicated concentrations of KGN. Controls received either culture medium only with 10% FBS. The impedance value of each well was monitored every hour and expressed as a cell index (CI) value. The experiments were performed in quadruplicates and impedance profiles were recorded continuously over nine days.

Cell Proliferation was assessed using the xCELLigence Real-Time Cell Analysis (RTCA) system (Roche, Switzerland) on Matrigel coated CIM-16 xCELLigence plates. The E-96 plate consists of incorporated gold cell sensor arrays which allow for the monitoring of cells inside each well. Electronic impedance of the sensors was measured through the detection of cells adhering to the electrodes. Cell attachment acts as insulation altering the electrode/solution interfaces, thereby increasing impedance. The E-96 plate was connected to the RTCA system and background impedance was measured. G52 and G53 cells were seeded onto the plate at an optimised density of 8 × 10³ cells per well. The plate was connected to the RTCA system and incubated at 37  °C. Cell adhesion, growth and proliferation were measured every 15 min for 48 h via the incorporated sensor electrode rays. Four replicates of the cell concentration were performed in each test. Electrical impedance was measured by the xCELLigence RTCA software as an arbitrary parameter labelled Cell Index (CI).

The migration capacity of cells was evaluated using the xCELLigence real-time cell analysis (RTCA) system (Roche, Penzberg, Germany) as previously described [20 (link)]. Briefly, 160 µL and 30 µL of media was added to the lower and upper chambers of modified 16-well plates (CIM-16, Roche), respectively. The lower chambers either contained FBS supplemented or FBS free medium to assess chemotactic and background migration. CIM-16 plates were subsequently placed in the RTCA DP instrument at 37 °C for 1 h to measure background signal. Serum-deprived stable MCF-7 cells were harvested using TrypLE ExpressTM (Invitrogen, Waltham, MA, USA), resuspended in serum-free medium, and seeded into the upper chambers of the CIM-16 plates at a density of 3 × 10⁴ cells/well. After adding the cells, CIM-16 plates were incubated in the laminar flow hood for 30 min at room temperature allowing cells to settle before placing them in the RTCA DP instrument at 37 °C. Chemotactic migration values were obtained by subtracting background migration signals. At each condition measurements were performed in triplicate and analyzed for 12 h.