Iodonitrotetrazolium chloride solution

The soft agar colony formation assay is used to detect cellular anchorage-independent growth in vitro, a trait of transformation ability. Briefly, 2% agar was melted and incubated in the water bath at 37 °C. Then the agar was diluted with complete culture media to get the 0.5% agar, which was added into 6-well plate (2 mL per well). Then the plates were placed at 4 °C for 30 min and 37 °C for another 30 min. During this period, the cells were counted and suspended in 0.4% agar. After the 0.5% agar beds had been ready, 1.5 mL 0.4% agar containing 4.5 × 10⁴ were layered onto the 0.5% agar bed in each well. After incubation at 4 °C for 30 min, the plates were cultured at 37 °C incubator for ~3 weeks with supplementing 1 mL 0.4% agar every week. Finally, the colonies were stained with 0.02% iodonitrotetrazolium chloride solution (Sigma-Aldrich, MO) and photographed for counting.

A total of 2 mL of 0.5% agar was added to six-well plates as the base layer, and 2.0 × 10⁴ cells were seeded in 0.4% top agar (1.5 mL per plate). In addition, 1 mL of medium with 0.4% agar was added to the top of each plate every five days for two weeks. The colonies were stained with 0.02% iodonitrotetrazolium chloride solution (Sigma-Aldrich, Saint Louis, MO, USA), photographed, and counted.