Evaluating Synergistic Effects of Drug Combinations

PC-3 cells were seeded on 96-well plates (2,000 cells/well in 100 µL), with three replicates used for each concentration. The cells were treated with various concentrations of sorafenib or clofoctol before incubation with 10 µL per well of Cell Counting Kit-8 (CCK-8) reagent (Dojindo Molecular Technologies Inc., Shanghai, China) or Alamar blue (Sigma-Aldrich, MO, USA) solution for 3 hours. Cell viability was estimated by CCK-8 assay or Alamar blue assay.21 (link) The OD was measured using a microplate reader (Thermo Fisher Scientific). The IC₅₀ values were calculated using the GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). The combined effect can be compared to the expected additive effect given by the common formula E_ab = E_a + (1-E_a) E_b, where E_ab represents the theoretical effect of the two drug combinations and Ea and Eb represent the two drugs.22 (link) To determine the potential mechanism of drug–drug interactions, we used the CompuSyn software (CompuSyn, Cambridge, UK) to calculate the combination index (CI). A CI of <1.0 was considered to be indicative of synergism.