Icelligence system

For the analysis of proliferation, the cells were seeded onto an E-Plate L8 within an iCELLigence system (ACEA Biosciences, San Diego, CA, USA), which had integrated microelectrode sensors in the bottom of the wells, and were incubated at 37 °C with 5% CO₂ for 150 h. In an iCELLigence system, as the cells proliferate, they adhere to the micro-electrodes, and alterations in electrical impedance reflect the biological status of the cells; therefore, the system allows for the monitoring of time-dependent changes of this parameter. Cell status is expressed as a normalized cell index; cell doubling rate was estimated.
Because of the non-functionality of their mitochondria, ρ0 cells produced large amounts of lactic acid by anaerobic glycolysis. Lactate levels in the culture medium were determined using a biochemical analyzer A25 (BioSystems, Barcelona, Spain) with an assay in which lactate was oxidized to pyruvate and hydrogen peroxide, which reacts with TOOS to form a colored compound. Lactate concentration was normalized for cell number.

The growth rate of cells with siRNA-induced gene knockdown was measured using the iCelligence system (ACEA Bioscience, CA, USA). Briefly, one day after transfection, cells were seeded in 8-well cell culture plates (1.5×10⁴ cells per well) and cellular impedance was measured every 2 hours for 96 hours. Cell index was an arbitrary measurement derived from electrical impedance that reflected the number of living cells. All experiments were repeated independently three times.

The iCELLigence system (ACEA Biosciences Inc., San Diego, CA, USA) was utilized to monitor cellular events in real time by recording the electrical impedance signal, followed by converting the impedance value into a cell index (CI) value. The CI is an arbitrary unit that reflects the cell number, morphology, and viability in a given culture well. 1 × 10⁴ myoblast cells were plated in each well of an E-plate L8 and further cultivated at 37°C in a humid atmosphere containing 5% CO₂. Seeding was allowed for 24 h followed by treatment with C. vulgaris and incubation for up to seven days. The CI value was recorded every 10 minutes, and the graph of myoblast cell proliferation was plotted using RTCA Data Analysis Software version 1 (ACEA Biosciences Inc., San Diego, CA, USA). Two E-plate L8 plates were run simultaneously for all dosages of C. vulgaris treatment, with two replicates (n = 2) for all treatment groups.