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Clonogenic assay of cells in vitro

Nature Protocols volume 1, pages 2315–2319 (2006)Cite this article

Abstract

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 defined 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 1–3 weeks. Colonies are fixed 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 dose–survival curves is included.

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Figure 1: Clonogenic assay performed in six-well plates, with clones produced by SW-1573 lung tumor cells.
Figure 2: Survival curves of Glioblastoma multiforma cells plated immediately (closed symbols) and delayed plated (open symbols) after irradiation.

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Acknowledgements

We thank Professor Dr. G.W. Barendsen for his invaluable contribution.

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Authors and Affiliations

  1. Department of Radiotherapy, Laboratory for Experimental Oncology and Radiobiology (LEXOR), University of Amsterdam, PO Box 22700, Amsterdam, 1100 DE, The Netherlands

    Nicolaas A P Franken, Hans M Rodermond, Jaap Haveman & Chris van Bree

  2. Department of Cell Biology and Histology, Centre for Microscopical Research, Academic Medical Centre, University of Amsterdam, PO Box 22700, Amsterdam, 1100 DE, The Netherlands

    Jan Stap

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  1. Nicolaas A P Franken
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  2. Hans M Rodermond
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Correspondence to Nicolaas A P Franken.

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Franken, N., Rodermond, H., Stap, J. et al. Clonogenic assay of cells in vitro. Nat Protoc 1, 2315–2319 (2006). https://doi.org/10.1038/nprot.2006.339

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