Xcelligence mp

xCELLigence MP (Roche Diagnostics) was used to monitor cell proliferation in real time. Background impedance was measured before seeding the cells using 40 µl of RPMI 1640 containing 10% FCS and always subtracted as blank value. 1 × 10⁴ cells in 50 µl of RPMI containing 10% FCS were added. Cell proliferation was measured with a programmed signal detection every 1 h and the signal was normalized to the transfection or palbociclib treatment time point where the cell index at every time point was divided by the cell index at the time of transfection. Data acquisition and analysis was performed with the RTCA software (version 1.2, Roche Diagnostics). IC50 values were defined as the drug concentration that inhibited cell proliferation by 50% at 48 h of treatment and were calculated using GraphPad Prism (version 6).

Three thousand Ewing sarcoma cells per well were seeded in a special 96-well plate (E-plate 96™, Roche Applied Science, Mannheim, Germany) and the proliferation index was recorded. Cell proliferation was evaluated at 30-min intervals on the basis of real-time impedance measurements performed with the xCELLigence system (xCELLigence MP™, Roche Applied Science, Mannheim, Germany). Experiments were performed in ten replicates and continued until the mock-treated control cells reached confluence. Dose-response-graphs and the resulting LD50s were calculated by analyzing 10 wells per dose according to the manufacturer’s recommendations.

Real-time label-free impedance-based monitoring of cellular proliferation assay was performed by using xCELLigence MP (Roche Applied Science). Transfected cells were incubated in 6-well plates for 48 h. After the incubation period, 5000 cells/well were seeded in E-plate 96. Cell proliferation was monitored at every 15 min for 72 h. Changes in proliferation rate were expressed as “cell index” (RTCA software 1.2.1, Roche Applied Science).