Anchorage-Independent Cell Growth Assay

Anchorage-independent growth was determined by a soft agar assay as described previously.24 (link) Briefly, a bottom layer of 1.4 mL (0.6%) was prepared with a 1:1 mixture of 1.2% low melting-point agarose (AMRESCO, OH, USA) (approximately 50°C) and warm 2 × DMEM, and was poured into each well of six-well plates. A top layer, which was a mixture of 500 μL of warm 2 × DMEM, 500 μL of the 0.6% base agar and cells at the density of 1 × 10³ cells per well, was poured on top of the solidified bottom layer. The cultures were fed 1 mL of DMEM supplemented with 10% FBS, which was gently refreshed twice a week. The plates were kept in a cell culture incubator maintained at 37°C under 5% CO₂ to allow colony growth. After 2 weeks of culture, the colony assay was terminated and cell colonies of more than 50 cells in a cluster were counted using a graduated eyepiece fitted in a transmission light microscope at a magnification of 40.25 (link)