Xcelligence dp device

MTS assay (Brand, Voerde-Friedrichsfeld, Germany) was used to measure the metabolic activity of cells: 5 × 10³ cells per well were seeded into 96 well plates and treated with 0–500 μM diacerein. The cells were treated for 24 h and 48 h, thereafter a CellTiter 96 AQueous Assay (Promega, Mannheim, Germany) was performed following the manufacturers’ instructions; untreated cells were used for control (ctrl).
The xCELLigence DP device from Roche Diagnostics (Mannheim, Germany) was used to monitor cell proliferation in realtime after cells were seeded on electronic microtiter plates (E-Plate; Roche Diagnostic) [16 (link)]. Cells were treated with 0, 30, 100, and 300 μM diacerein and the proliferation rate was measured for 24 h. Cell index (CI) measurements were performed in triplicates with a signal detection set for every 20 min. The cell index (CI) is a measure for the cell density of cells and was normalized to the time point when diacerein was added. Subsequent to the continuous xCELLigence cell monitoring, the slope (1/h) representing the rate of change of the cell index was calculated from time 7–24 h. Acquisition and analysis was performed with the RTCA software (Version 1.2, Roche Diagnostics).

The xCELLigence DP device from Roche Diagnostics (Mannheim, Germany) was used to monitor cell proliferation in real-time. This system allows the measurement of the impedance in real-time; the higher the value of the impedance, the higher the amount of vital cells growing on the surface of each well; a maximal value is reached when the cell layer becomes confluent. After ultrasonic irradiation with 24.5 kHz, 5 × 10³ or 1 × 10⁴ cells were seeded per well in quadruplicates in electronic microtiter plates (E-Plate; Roche Diagnostic) and measured with programmed signal detection every 20 min for 92 h with the xCELLigence system according to the manufacturer’s instructions. Data acquisition and analyses were performed with the RTCA software (version 1.2, Roche Diagnostics).

The xCELLigence DP device (Roche Diagnostics, Germany) was used to monitor cell proliferation in real-time. The 96 well electronic microtiter plates (Roche Diagnostics, Germany) were used to culture H292 cells (5 × 10⁵), and the cells were treated with various concentrations of 0404 or Nutlin or 5-FU, respectively. The xCELLigence system was used to measure all cells for 96 h according to the instructions. A programmed signal detection was used to measure the cell density in quadruplicate every 30 min. The RTCA software (version 1.2) from Roche Diagnostics was used for collecting and analyzing data.

An xCELLigence DP device (Roche Diagnostics, Germany) was used to monitor cell proliferation in real time. HCC cells (5 × 10⁵) were seeded in 96-well electronic microtiter plates (Roche Diagnostics, Germany) and then were treated with Dox combined with AMSC-Exo-199a or AMSC-Exo-67. The xCELLigence system was used to measure all cells for 96 h according to the instructions. A programmed signal detector was used to measure the cell density in quadruplicate every 30 min. RTCA software (version 1.2) from Roche Diagnostics was used for collecting and analyzing data. For the 50% inhibitory concentration (IC50) assay, data were analyzed by GraphPad Prism 5 software (GraphPad, La Jolla, CA).

Using the xCELLigence DP device from Roche Diagnostics, real-time measurements of cell migration on MDA-MB-231 cells were performed. Cells were seeded at 30,000 per well in CIM-Plates 16 (Roche Diagnostics, Risch-Rotkreuz, Switzerland) in serum-free medium in the presence or absence of inhibitors. Full growth medium was used as a chemo-attractant in the lower chamber. As cells pass through the 8 µm pores towards the chemo-attractant, they adhere to the underside of the filter, embedded with a gold microelectrode. This produces an electrical impedance signal, which correlates with the number of migrating cells. Cell index is an arbitrary unit based upon the measured cell-electrode impedance derived by the software using the following calculation as described in reference [34 (link)].

The cytotoxicity of MEAN on Huh7.5.1 cells was evaluated using real‐time cellular analysis (RTCA). Briefly, the xCELLigence DP device from Roche Diagnostics (Mannheim, Germany) was used to monitor the cell cytotoxicity in real‐time. Huh7.5.1 cells (5 × 10³) were seeded in 96‐well electronic microtitre plates (E‐Plate^™; Roche Diagnostics) and cultured with DMEM containing 0, 0.31, 0.63, 1.25, 2.5, 5, 10 or 20 μM of MEAN. Cell density measurements were performed in quadruplicate with a programmed signal detection every 20 min. Data acquisition and analyses were performed with the RTCA software (version 1.2; Roche Diagnostics).
The cytotoxicity of IFN‐α or RBV (Sigma‐Aldrich) was determined using the cell counting kit 8 (CCK‐8) assay (Dojindo, Kumamoto, Japan). Briefly, Huh7.5.1 cells (5 × 10³) were seeded into 96‐well culture plates and then treated with various concentrations of IFN‐α (0, 312.5, 625, 1250, 2500, 5000, 10,000 U/ml) or RBV (0, 2.13, 4.25, 8.5, 17, 34, 68 μM) for 48 hrs. Next, the treated cells were incubated with CCK‐8 solution (1/10 vol/vol in serum‐free media) for an additional 3 hrs at 37°C. The absorbance was determined at 450 nm using a microtitre plate reader (Bio‐Rad, Hercules, CA, USA).