2.2. 4T1 Cell Culture and In Vitro Assays

The 4T1 breast cancer cell line was purchased from American Type Culture Collection (ATCC). The 4T1 cells were maintained in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 100 U/mL of penicillin and 100 U/ml of streptomycin (Gibco, Carlsbad, CA, USA) at 37 °C in an incubator containing 5% CO₂.

CCK8 assay (Biosharp, Hefei, China) was used to study the cell viability. A total of 1 × 10⁴ cells were seeded into a 96-well plate in 100 μL RPMI-1640 medium. For detection, 10 μL CCK8 reagent was added into each well and absorbance at 450 nm was recorded after 1-h incubation in dark.

Wound healing assay and transwell assay were applied according to the protocols used in our previous studies [18,19]. Wound healing assay was used to assess the migration ability of 4T1 cells. Briefly, 5 × 10⁵ cells were seeded into a 6-well plate and treated with different concentrations of EGCG. 4T1 cells were scratched with a sterile 200 μL tip and monitored for the following 48 h. Representative images were taken at 24 h and 48 h after scratching. Tranwell assay was applied to detect cell invasion ability. Transwell chambers (Corning, Rochester, NY, USA) were put into a 24-well transwell plate. Twenty percent FBS in cell medium was added into the lower chamber, and 5 × 10⁵ 4T1 cells were seeded in the upper well. After treatment with different EGCG for 24 h, the images on the lower side of the insert filter were taken under microscope. The invasion ability (relative invasion percentage) was calculated by counting three different fields of cells adhering to the lower surface of the transwell system.