ColonyArea: an ImageJ plugin to automatically quantify colony formation in clonogenic assays

PLoS One. 2014 Mar 19;9(3):e92444. doi: 10.1371/journal.pone.0092444. eCollection 2014.


The clonogenic or colony formation assay is a widely used method to study the number and size of cancer cell colonies that remain after irradiation or cytotoxic agent administration and serves as a measure for the anti-proliferative effect of these treatments. Alternatively, this assay is used to quantitate the transforming potential of cancer associated genes and chemical agents. Therefore, there is a need for a simplified and standardized analysis of colony formation assays for both routine laboratory use and for parallelized automated analysis. Here we describe the freely available ImageJ-plugin "ColonyArea", which is optimized for rapid and quantitative analysis of focus formation assays conducted in 6- to 24-well dishes. ColonyArea processes image data of multi-well dishes, by separating, concentrically cropping and background correcting well images individually, before colony formation is quantitated. Instead of counting the number of colonies, ColonyArea determines the percentage of area covered by crystal violet stained cell colonies, also taking the intensity of the staining and therefore cell density into account. We demonstrate that these parameters alone or in combination allow for robust quantification of IC50 values of the cytotoxic effect of two staurosporines, UCN-01 and staurosporine (STS) on human glioblastoma cells (T98G). The relation between the potencies of the two compounds compared very well with that obtained from an absorbance based method to quantify colony growth and to published data. The ColonyArea ImageJ plugin provides a simple and efficient analysis routine to quantitate assay data of one of the most commonly used cellular assays. The bundle is freely available for download as supporting information. We expect that ColonyArea will be of broad utility for cancer biologists, as well as clinical radiation scientists.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Colony-Forming Units Assay / methods*
  • Humans
  • Staurosporine / analogs & derivatives
  • Staurosporine / pharmacology
  • Tumor Cells, Cultured


  • 7-hydroxystaurosporine
  • Staurosporine

Grant support

This work was supported by the Academy of Finland fellowship grant, the Sigrid Juselius Foundation, the Cancer Society of Finland and the Marie-Curie Reintegration Grant to DA. JW was supported by funding from the Foundation of the Finnish Cancer Institute and the Sigrid Juselius Foundation. The funders had no role in study design, data collection and analysis decision to publish, or preparation of the manuscript.