4.4. Real-Time Monitoring of Cytotoxicity

This assay utilized the xCELLigence^® real-time cells analyzer to monitor the effects of the compounds to the cells. Cell titration was first conducted to determine the appropriate seeding density in the assay (Figure S1). To start the real-time cell analysis, background readings from 100 μL of media added in each well of the E-plate^® 96 was obtained. A 100 μL aliquot of the cell suspension (2 × 10⁴ cells/well) was added, and the plate was equilibrated at RT for 30 min before placing in the RTCA-SP station inside the 5% CO₂ incubator. After ~24 h, the cells were treated with the compounds in the same manner as in the MTT cytotoxicity assay. Cellular responses were monitored every 30 s for 1 h, then every 2 min for another 1 h, and finally every 30 min for 144–168 h (six to seven days). Cell index values were normalized to the time of addition of compounds and plotted in GraphPad PrismTM version 3.03 (San Diego, CA, USA). IC₅₀s were calculated using the sigmoidal-dose response curve-variable slope equation using the RTCA-SP software version 2.0 (San Diego, CA, USA).