4.8. Soft Agar Colony Formation Assay

The base agar containing 0.5% low-melting-temperature agarose (Sigma-Aldrich, St. Louis, MO, USA) in 2× cell culture medium was seeded and coated on 12-well plates and incubated for 30 min at 4 °C. Huh7 cells (4 × 10³ cells/well) were suspended in cell culture medium containing 0.3% low-melting-temperature agarose and plated on a semisolid 0.5% agarose medium. The cell culture medium containing GCB was freshly replaced every 3 days. After incubation for 14 days at 37 °C under 5% CO₂, the colonies were stained with 0.05% crystal violet solution for 1 h at 25 °C and washed several times with PBS. The stained samples were photographed using a digital camera (Olympus SP-350, Cam2Com).