Experiments were carried out using the xCELLigence RTCA DP instrument (Roche Diagnostics GmbH, Mannheim, Germany) which was placed in a humidified incubator at 37°C and 5% CO2.
Cell proliferation and cytotoxicity experiments were performed using modified 16-well plates (E-plate, Roche Diagnostics GmbH, Mannheim, Germany). Microelectrodes were attached at the bottom of the wells for impedance-based detection of attachment, spreading and proliferation of the cells. Initially, 100 µL of cell-free growth medium (10% FBS) was added to the wells. After leaving the devices at room temperature for 30 min, the background impedance for each well was measured. Cells were harvested from exponential phase cultures by a standardized detachment procedure using 0.05% Trypsin-EDTA (Invitrogen NV/SA, Merelbeke, Belgium) and counted automatically with a Scepter 2.0 device (Merck Millipore SA/NV, Overijse, Belgium), Fifty µL of the cell suspension was seeded into the wells (20, 40, 80, 100, 200, 400 and 800 cells/well for proliferation, 1000 cells/well for cytotoxicity experiments). The cell concentrations of 20, 100, 200 and 400 cells/well were considered for correlation with the SRB method described below. After leaving the plates at room temperature for 30 min to allow cell attachment, in accordance with the manufacturer's guidelines, they were locked in the RTCA DP device in the incubator and the impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index value (CI). Water was added to the space surrounding the wells of the E-plate to avoid interference from evaporation. For proliferation assays, the cells were incubated during ten days in growth medium (10% FBS) and CI was monitored every 15 min during the first six hours, and every hour for the rest of the period. Two replicates of each cell concentration were used in each test. For cytotoxicity experiments, CI of each well was automatically monitored with the xCELLigence system every 15 min during the overnight recovery period. Twenty-four hours after cell seeding, cells were treated during a period of 72 hours with paclitaxel (0, 1, 2, 5, 10, 20, 50 and 100 nM) dissolved in phosphate buffered saline (PBS). PBS alone was added to control wells. Each concentration was tested in duplicate within the same experiment. CI was monitored every 15 min during the experiment. Three days after the start of treatment with paclitaxel, CI measurement was ended.
Cell proliferation and cytotoxicity experiments were performed using modified 16-well plates (E-plate, Roche Diagnostics GmbH, Mannheim, Germany). Microelectrodes were attached at the bottom of the wells for impedance-based detection of attachment, spreading and proliferation of the cells. Initially, 100 µL of cell-free growth medium (10% FBS) was added to the wells. After leaving the devices at room temperature for 30 min, the background impedance for each well was measured. Cells were harvested from exponential phase cultures by a standardized detachment procedure using 0.05% Trypsin-EDTA (Invitrogen NV/SA, Merelbeke, Belgium) and counted automatically with a Scepter 2.0 device (Merck Millipore SA/NV, Overijse, Belgium), Fifty µL of the cell suspension was seeded into the wells (20, 40, 80, 100, 200, 400 and 800 cells/well for proliferation, 1000 cells/well for cytotoxicity experiments). The cell concentrations of 20, 100, 200 and 400 cells/well were considered for correlation with the SRB method described below. After leaving the plates at room temperature for 30 min to allow cell attachment, in accordance with the manufacturer's guidelines, they were locked in the RTCA DP device in the incubator and the impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index value (CI). Water was added to the space surrounding the wells of the E-plate to avoid interference from evaporation. For proliferation assays, the cells were incubated during ten days in growth medium (10% FBS) and CI was monitored every 15 min during the first six hours, and every hour for the rest of the period. Two replicates of each cell concentration were used in each test. For cytotoxicity experiments, CI of each well was automatically monitored with the xCELLigence system every 15 min during the overnight recovery period. Twenty-four hours after cell seeding, cells were treated during a period of 72 hours with paclitaxel (0, 1, 2, 5, 10, 20, 50 and 100 nM) dissolved in phosphate buffered saline (PBS). PBS alone was added to control wells. Each concentration was tested in duplicate within the same experiment. CI was monitored every 15 min during the experiment. Three days after the start of treatment with paclitaxel, CI measurement was ended.
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