In Vitro Tumorigenic Assay: Colony Forming Assay for Cancer Stem Cells

Colony forming or clonogenic assay is an in vitro quantitative technique to examine the capability of a single cell to grow into a large colony through clonal expansion. Clonogenic activity is a sensitive indicator of undifferentiated cancer stem cells. Here, we described the colony forming ability of the isolated breast cancer stem cells from the total population of cancer cells using double-layered, soft agarose-based assay. This method demonstrates that cancer stem cells can survive and generate colony growth in an anchorage-independent culture model. The 0.005% crystal violet solution is used in this assay to visualize the generated colonies.