Rtca dp

Real-time cell analysis (RTCA; Roche Applied Science, GmbH, Penzberg, Germany) experiments were carried out using the xCELLigence RTCA DP instrument. Briefly, 5 × 10³ cells were seeded in 16-well microtiter E-plates for the cell proliferation assay, and after incubating for 15 min, the baseline was detected. After the cells were incubated for 10 hours, FO (2.0 mM) and DMSO were added to the Huh7 and Hep3B cells, respectively, and then the electrical impedance in each well was measured continuously for nearly 90 hours. The slope indicates the increase in cell impedance per unit time (slope = impedance/time). The mean ± SD was calculated from three individual replicate wells.

Real time cell proliferation experiments were performed using the RTCA DP instrument (Roche Diagnostics GmbH), which was placed in a humidified incubator maintained at 5 % CO₂ and 37 °C. For proliferation assays, cells were seeded in complete medium in 16-well plates (E-plate 16, Roche Diagnostics GmbH) at a density of 2000 cells/well after coating with 10 μg/mm² fibronectin (Sigma Biochemicals). The plate containing gold microelectrodes on its bottom was monitored every 10 min for 4 h (adhesion process), then once every 30 min, until the end of experiment, for a total of 72 h. Data analysis was performed using RTCA software 1.2 supplied with the instrument.

Six treatment groups of cells were used [Table S1(ii) and illustrated in Figure 1A‐F]. For the intercellular direct contact experiment (ICDBMSC), HUVEC were separated from DBMSCs by transwell chamber membrane culture system [Catalogue number 657640, Greiner Bio‐One].⁹ HUVEC were harvested using TrypLE™ solution (Life Technologies) and then used in the adhesion and proliferation experiments.
The adhesion and proliferation of HUVEC were evaluated using the xCELLigence system (RTCA‐DP; Roche Diagnostics) as previously described.⁹, ¹⁰, ¹³ Briefly, 2 × 10⁴ HUVEC [Table S1(ii) and illustrated in Figure 1A‐F] were cultured in complete endothelial cell growth medium in quadruplicate wells of 16‐well culture E‐Plates (Catalogue number 05469813001, Roche Diagnostics), and the proliferation index (mean ± standard errors) was measured as previously described.⁹, ¹⁰, ¹³ For cell adhesion, data were measured at 2 hours, whereas the rate of cell proliferation was determined by calculating the normalized cell index at 24, 48 and 72 hours after the adhesion time‐point (2 hours). Each experiment was performed and repeated as described above.