Rhodanile blue

One-hundred, 500 or 1000 keratinocytes were plated on a 3T3 feeder layer per 10 cm diameter dish or per well of a six-well dish. After 12 days, feeders were removed and keratinocyte colonies were fixed in 10% formalin (Sigma) for 10 min then stained with 1% Rhodanile Blue (1:1 mixture of Rhodamine B and Nile Blue A [Acros Organics]). Colonies were scored using ImageJ and clonogenicity was calculated as the percentage of plated cells that formed colonies.

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)}.

100 or 400–800 live (Trypan Blue-negative) NHK or SCC13 cells, respectively, were seeded per condition into duplicate or triplicate wells (containing a 3T3-J2 feeder layer) of a six-well dish (Falcon). After 12 days, feeder cells were completely removed by rinsing with PBS and colonies were either fixed in 4% (w/v) paraformaldehyde for 10 min and then stained with 1% Rhodanile Blue (1:1 mixture of Rhodamine B and Nile Blue A (Acros Organics))12 (link), or simultaneously fixed and stained with Crystal Violet solution (0.4% (w/v) crystal violet, 20% (v/v) methanol). Colonies were imaged and counted using an automated cell colony counter (Gelcount, Oxford Optronix, UK), and CFE was calculated as the average percentage of seeded cells that formed colonies. Colony area was measured using the Fiji image processing software package and the ‘Analyse Particles' tools, with a minimum particle size of 0.01 mm².