*4.4. MTT Assay*

Stock solutions of MAW, MAE, and DOX were dissolved in dimethyl sulfoxide (DMSO) at the concentration of 50 mg/mL (extracts) or 1 mM (DOX). Cells were seeded into 96-well microtiter plates at a density of 5000 cells/well. After 24 h, they were treated with five di fferent extract concentrations (12.5, 25, 50, 100, and 200 μg/mL) or DOX (0.31, 0.62, 1.25, 2.5, and 5 μM). To determine the combined effect of DOX and extracts, cells were treated with various concentrations of DOX in the presence of subtoxic concentrations of MAW or MAE (5, 10, or 20 μg/mL). The control cells were grown in culture medium only. After an additional 72 h of incubation, cell survival was determined by MTT test, as described elsewhere [44–46]. The absorbance was measured at 570 nm using Multiskan EX reader (Thermo Labsystems Beverly, MA, USA). The experiments were performed in triplicate, and the data are presented as mean ± standard deviation (SD) of the results obtained in three independent experiments.

Combination index (CI) was used to determine the degree of interaction between DOX and *M. aquifolium*, and its formula is the sum of the ratio of the dose of each drug in the compound to the dose when used alone when the combination and compound produce 50% e fficacy [47].

The CI values represent the mean of three experiments with the following values: CI 1.3: antagonism; CI 1.1–1.3: moderate antagonism; CI 0.9–1.1: additive e ffect; CI 0.8–0.9: slight synergism; CI 0.6–0.8: moderate synergism; CI 0.4–0.6: synergism; and CI 0.2–0.4: strong synergism [48,49].
