*2.4. Cell Viability*

The MTT (3-(4,5-dimethylthiazol-2-yl) assay was employed to evaluate the effect of mistletoe extract on the viability of MDA-MB-231 cells and THP-1 cells. The MTT reagent used for this assay was procured from Duchefa Biochemie (Haarlem, Netherlands). For the MDA-MB-231 cells, a concentration of 1 × 104 cells/well was added to 96-well plates (SPL Life Sciences Co., Ltd., Pocheon-si, Republic of Korea) and subsequently cultured at 37 ◦C for 24 h within a humidified 5% CO2 incubator (Sanyo) without mistletoe extract. After removing the supernatant, mistletoe water extract was introduced, and the cells were further cultured for either 24 h or 48 h. In the case of THP-1 cells, differentiation into macrophages was achieved by treating the cells with phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA). The differentiated macrophages were then distributed into a 100 mm cell culture dish and incubated at 37 ◦C for 48 h within a humidified 5% CO2 incubator (Sanyo). Afterward, the differentiated macrophages were washed with phosphate-buffered saline (PBS, VWR Life Science, Radnor, PA, USA), followed by an additional 48 h incubation with the same medium. The cells were subsequently seeded into a 96-well plate at a concentration of 1 × <sup>10</sup><sup>4</sup> cells/well and exposed to various concentrations (0.1–500 μg/mL) of mistletoe extract. To measure cell viability, 50 μL of MTT solution (5 mg/mL) prepared in PBS was added to each well and allowed to incubate at 37 ◦C for 4 h. After the supernatant was removed and the formazan crystals were dissolved using with 200 μL of dimethyl sulfoxide (DMSO), absorbance was assessed at 570 nm using a microplate reader (Sunrise Technologies, Männedorf, Zürich, Switzerland).
