Soft Agar Assay

The soft agar colony formation assay is a traditional method to determine the anchorage-independent growth in vitro, which is considered the most stringent assay for detecting the malignance of cells. Briefly, we melt 1.4% (v/v) agarose (Sigma-Aldrich) in a microwave and cooled to room temperature. Then, equal volumes of the 1.4% melted agarose were mixed with the complete cell culture medium. After that, 2 ml of 0.7% (v/v) low-melting point agar was added into each well of the six-well plate and set aside to allow agarose to solidify. Cells (5,000/ well) were mixed with 1.4% agarose in complete culture medium, plated on top of the solidified layer to form colonies in 1–3 wk. Cells were fed with complete culture medium every 3 d.

The colony is defined to consist of at least 50 cells¹³. After the cells have formed sufficiently large clones, we removed the medium above the cells and rinsed carefully with PBS. Then, colonies were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min and stained with crystal violet solution (0.5%, v/v) (Sigma-Aldrich) for at least 30 min. After removal of the crystal violet solution and carefully washing with tap water until excess dye was removed, the plates were left to dry at room temperature. Colonies were counted manually by light microscopy.