Quantification of Keratinocyte Colony Formation

100 live (Trypan Blue-negative) human keratinocytes were seeded per condition into triplicate wells (containing a 3T3-J2 feeder layer) of a 6-well dish (Falcon). After 12 days, feeder cells were removed by rinsing with EDTA and colonies were either fixed in 4% (w/v) paraformaldehyde for 10 min and stained with 1% Rhodanile Blue (1:1 mixture of Rhodamine B and Nile Blue A (Acros Organics))^{22 (link)}, or simultaneously fixed and stained with Crystal Violet solution (0.4% (w/v) crystal violet, 20% (v/v) methanol)^{35 (link)}. Colonies were imaged and counted using an automated cell colony counter (Gelcount™, Oxford Optronix, UK), and colony forming efficiency (CFE) was calculated as the average percentage of seeded cells that formed colonies^{35 (link)}. Colony area was measured using the Fiji image processing software package and the “Analyze Particles” tools, with a minimum particle size of 0.01 mm^{2 35 (link)}. Colonies were scored as abortive if they contained fewer than 40 cells, the majority of the cells being large and terminally differentiated^{43 (link)}.