2.3. Detection of Acacetin Cytotoxicity

Cells in logarithmic growth phase were prepared into cell suspensions that were subsequently inoculated into 96-well plates. To seed approximately 1–2 × 10⁴ cells per well, approximately 100 μl of cell suspension was added into wells; one sample generated 4–6 replicates. Plates were then placed in a 37°C incubator to allow the cells to stably adhere. Subsequently, cells were incubated a gradient of acacetin concentrations (1.56, 3.13, 6.25, 12.50, 25.00, 50.00, 100.00, and 200.00 μg/ml) for 12, 24, and 36 h to observe cell growth. Cell Counting Kit 8 (CCK-8; Wuhan Boster Biotechnology, Wuhan, China) reagents were nontoxic to cells and reduced by the electron carrier to orange-deficient formazan by some dehydrogenases in the mitochondria of cells. Cell viability assay is according to a previously described protocol [16].