Keratinocyte Colony Formation Assay: An In Vitro Assay to Assess the Ability of Keratinocytes to Grow into Colonies

- Colony formation assay determines the ability of individual cells to proliferate and form colonies. Begin with an excised mouse-dorsal skin in a culture plate. Scrape off the subcutaneous tissue and fat from the ventral side of the skin. Treat the tissue with a digestion solution. The proteolytic enzymes in the solution degrade the extracellular matrix, inducing epidermal cell dissociation.

Gently scrape the epidermal layer to release the dissociating cells and hairs into a harvesting media. Filter the suspension through an appropriate strainer to trap hairs or any debris and obtain a single-cell suspension of epidermal cells. Transfer the desired volume of this cell suspension into a collagen-coated plate containing an adherent layer of growth-arrested feeder cells. Supplement with a suitable media that favors keratinocyte survival.

During culture, the keratinocytes attach to the feeder layer. Additionally, these feeder cells secrete growth factors and synthesize extracellular matrix proteins that support the growth of keratinocytes. Depending on the proliferative capability of individual keratinocytes, they start forming colonies. Remove the media and fix the colonies. Stain th