2.7. Assaying anchorage‐independent growth in soft agar

Soft‐agar assays were performed to evaluate anchorage‐independent growth of cancer cells. Three milliliters of 0.6% bottom agarose were placed in separate wells of 6‐well plates with or without arctiin, arctigenin, Arctii Fructus extract, or bioconversion samples. After solidification of the bottom agarose, 2.4 × 10⁴ cells were resuspended in 0.3% top agarose, with or without sample, and 1 ml of the top agarose containing 8 × 10³ cells was placed on the bottom agarose. The cells were incubated at 37°C in a 5% CO₂ incubator for 3 weeks. The colonies were detected using an Eclipse Ti‐S microscope (Nikon) and photographed using a MetaMorph software program (Molecular Devices).