Xcelligence rtca system

Cytotoxic effect of MBIC was monitored in a time-dependent manner by using xCELLigence RTCA system (Roche, Manheim, Germany). 50 μl/well of cell-free completed medium was added in 96-well gold-microelectrode array (E-plate) to perform background measurements. 5 × 10³ cell/well were seeded and incubated at room temperature for 30 min. The E-plate was incubated in an E-plate 96 RTCA SP station at 37°C in 5 % CO₂ incubator overnight. Cell adherence, proliferation and spreading were recorded every 5 min and the cell sensor impedance was displayed as an arbitrary unit called normalized Cell Index (nCI). When the cells reached the stage of logarithmic growth, they were treated with various concentrations of MBIC (0.3, 0.7, 1.5 and 3 μM for MCF-7; 10, 20, 40 and 80 μM for MDA-MB-231) and were monitored continuously. To reduce variation between wells, CI values were normalized to the value at the beginning of the treatment time-point. Each experiment was performed in triplicate.

Cell migration and invasion were determined using the xCELLigence RTCA system (Roche). Cells were transfected with the indicated siRNAs for 48 h and starved in a medium without FCS 24 h before the start of the real-time recording. Cells were seeded in 16-well CIM plates (Roche) at the following densities: UM-UC-6 and UM-UC-10 (both at 50,000 cells/well); HT-1376, TCC-SUP-G, JON, and RT-112 (all at 60,000 cells/well); J82 (70,000 cells/well); and CAL-29 (80,000 cells/well). For the cell migration experiments, the membrane between the upper and lower chambers of the CIM plate was left uncoated, whereas for the cell invasion assays, it was coated with 20 µL of Matrigel (Corning (Glendale, AZ, USA)). Matrigel was diluted in a serum-free medium to a concentration of 400 µg/mL and allowed to solidify at 37 °C for 4 h before coating. FCS (10%) was used as a chemoattractant. Cell indices were recorded every 15 min.

Twenty-four hours after transfection, cells were seeded at 5 × 10³ cells/well in cell culture E-Plate 16 (ACEA Biosciences Inc., USA) for proliferation assays and at 2 × 10⁴ cells/well in cell culture CIM-Plate 16 (ACEA Biosciences Inc., USA) for migration assays according to the manufacturer’s instructions. The plates were incubated at 37 °C with 5% CO₂. The cell growth and migration curves were automatically recorded on the xCELLigence RTCA System (Roche, USA) in real-time. The cell index of the proliferation and migration assays was followed for 4–5 days.

The effect of SCIC2.1 on HepG2 cell was investigated using xCELLigence RTCA system (Roche) as a simple, yet quantitative, method to monitor changes in cell proliferation. This technology measures the flow of electrons between gold microelectrodes in cell plate. The number of adhering cells alters the electron transport and consequently, the impedance. Impedance is than expressed as an arbitrary unit parameter, “Cell Index” (CI) that is related to cellular density and is therefore, an indirect way to evaluate cell confluence. SCIC2.1 was used at 10 μM and 25 μM, and DMSO as a control, and dynamic CI values were monitored at 30-min intervals for 70 h. Calculated CI values were plotted on a line graph. Each experimental condition was tested in triplicate. The reported values correspond to the mean values corrected for the standard deviation.

Real-time cell behavior was monitored using xCELLigence RTCA system (Roche), which allows label-free and dynamic monitoring of cells by measuring electrical impedance. 10⁵ cells were seeded in 96 E-plates (Roche) 24 hrs prior 5-FU treatments. RTCA system displays the measurements of impedance signal as Cell Index (CI) values, providing quantitative information about the different biological status of the cells including number, viability, proliferation and mobility. CI values curves were normalized to the time point of 5-FU administration. MTS were performed at different time points on 10⁵ cells seeded into 96 well-plate 24 hrs prior 5-FU treatments using Cell Titer Aqueous One Solution Cell Proliferation assays as described by the manufacturer (Promega). Cell viability and total cell numbers were quantified in response to 5-FU treatment by trypan blue staining method using Cedex XS analyzer (Roche) from 150.10⁵ cells seeded in 24 well-plates 24 hrs prior 5-FU treatment.

Cell proliferation was determined using the xCELLigence RTCA system (Roche, Basle, Suizerland). Real-time recording of cell proliferation was started 24 h after transfection with the indicated siRNAs. In EGFR inhibition assays, cells were seeded in a medium containing the indicated concentration of Erlotinib HCI (OSI-774 HCI, Selleck Chemicals GmbH (Cologne, Germany), #S1023). Cells were seeded in 16-well E-plates (Roche) at the following densities: VM-CUB-1 (2500 cells/well); 5637, CAL-29, HT-1376, UM-UC-6, UM-UC-10, T24, TCC-SUP-G, J82, and RT-112 cells (all at 5000 cells/well); UM-UC-3 (7500 cells/well); and JON (10,000 cells/well). The system recorded cell indices at 15 min intervals.

Chondrocytes from two donors were plated in E-plate VIEW 16 (Cat. no. 06324746001, ACEA Biosciences) at 3000 cells per well in 100 μL DMEM, supplemented with 10% FBS and vehicle, 25(OH)D₃ or1α,25(OH)₂D₃ at 10^-6 M for 80 h and real-time proliferation was monitored using the xCELLigence RTCA system (Roche Diagnostics). The cell-index was normalized to 25 h (1 h after treatment) and log 2 converted to more accurately display growth rate [18 (link)].