In vitro cell proliferation assay

The Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was used to measure cell proliferation. Cells in logarithmic growth phase were collected from each group, and cell suspensions were added to a 96-well plate (100 µl per well) at approximately 9 × 10³, 5 × 10³, and 3 × 10³ cells per well for each group, respectively, followed by incubating the plates for 24, 48, and 72 h, respectively at 37 °C with 5% CO₂. Freshly prepared CCK-8 solutions (10 µl) were subsequently added to the plate and incubate for 1-4 h at 37 °C. For each group, six duplicate wells were used. An ELISA analyzer was then used to measure the absorbance (OD) of each well at 450 nm, and a cell growth curve was plotted. All experiments were performed in triplicate.