The majority of our lab protocols are based on those in Jeff Rosen’s Lab at the Baylor College of Medicine.
We have adapted the standard monolayer clonogenic assay done in standard monolayer culture to 3D stem cell promoting culture in non-adherent growth factor enriched culture described by Dontu et al, Genes Dev. 2003 May 15;17(10):1253-70 (Woodward and Bristow, Semin Radiat Oncol. 2009 Apr;19(2):87-95).
The potential value of incorporating stem cell–promoting culture conditions into clonogenic assays. Clonogenic assays are performed by digesting tumors or trypsinizing cell lines to isolate single cells.
In figure 1 above, various single cells (multi-potent and terminally differentiated) are highlighted in the center area (shaded light pink). Possible clonogenic colonies derived from each type of single cell are highlighted in blue boxes. On the left are in vitro colonies that would be generated from cells surviving radiation if the culture conditions (standard monolayer) did not promote the survival or expansion of the stem or progenitor cells. In this case, all colonies would represent differentiated cells capable of multiple mitoses after plating/radiation. This would provide no information about the radioresistance of the stem/progenitor population. On the right are colonies that would be generated in vitro from cells surviving radiation if the culture conditions (3D, serum-free, growth factor enriched) promoted the survival or expansion of the stem or progenitor cells and differentiated cells. In this case, colonies would represent differentiated cells capable of multiple mitoses after plating/radiation and colonies from the stem/progenitor population. Based on this model, measuring the effect of radiation on the stem/progenitor population would be possible only in culture conditions that promote stem/progenitor cells in addition to, or instead of, the differentiated cells.