2.6. Cell Viability Test and Calculation of the Combination Index

The stock of momelotinib and sorafenib was prepared by dissolving 20 mg/mL of the mixture in DMSO. The stocks of each drug were stored at −20 °C until use. Using the CompuSyn software to calculate the half-maximal inhibitory concentration (IC₅₀) values of different cell lines as previously reported by Chou TC and Martin N. The calculation method of IC₅₀ is as described in the PC Software and User’s Guide on the ComboSyn Inc. website (http://www.combosyn.com, accessed on 4 May 2020). The effects of momelotinib and sorafenib on cell proliferation were detected using the sulforhodamine B (SRB) assay. The synergistic effect of these two drugs was analyzed using isobolograms of the drug combination, as previously reported by Chou and Talalay [22,23]. The interaction between the two drugs was also analyzed by the median-effect principle proposed by them. The combination index (CI) was calculated using CompuSyn software. A CI value is less than 1 represented synergism [23]. Briefly, HCC cells or tumorspheres were seeded in 96-well plates (8 × 10⁵ cells/well) and treated with the drugs (momelotinib or sorafenib alone or in combination) at the 5 μM and 2.5 μM concentration for 48 h, respectively. After respective drug treatments, the relative cell number was estimated by the SRB reagent according to the manufacturer’s protocol (Sigma, Vallejo, CA, USA).