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PROTOCOL
Clonogenic assay of cells in vitro
Nicolaas A P Franken1, Hans M Rodermond1, Jan Stap2, Jaap Haveman1 & Chris van Bree1
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1Laboratory for Experimental Oncology and Radiobiology (LEXOR), Department of Radiotherapy, 2Department of Cell Biology and Histology, Centre for Microscopical Research, Academic Medical Centre, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands. Correspondence should be addressed to N.A.P.F. ([email protected]).
Published online 21 December 2006; doi:10.1038/nprot.2006.339
Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is dened to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo unlimited division. Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retains the capacity to produce colonies. Before or after treatment, cells are seeded out in appropriate dilutions to form colonies in 13 weeks. Colonies are xed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a stereomicroscope.
A method for the analysis of radiation dosesurvival curves is included.
INTRODUCTIONIn 1956, Puck and Marcus1 published a seminal paper describing a cell culture technique for assessment of the clone- or colony-forming ability of single mammalian cells plated in culture dishes with a suitable medium. The selected medium was supplemented with a large number of heavily irradiated feeder cells, which served to condition the microenvironment of the much smaller number of cells to be tested for their clone formation. The authors carried out experiments that yielded the rst radiationdose survival curve for HeLa cells in culture irradiated with X-rays.They showed that these mammalian cells were much more radio-sensitive than assumed earlier for cells in tissues, with mean lethal doses in the range of 12 Gy.
This clonogenic assay has been used in the ensuing decades for a large variety of studies with many types of...