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Analysis of clonogenic growth in vitro

Nature Protocols volume 16, pages 4963–4991 (2021)Cite this article

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Abstract

The clonogenic assay measures the capacity of single cells to form colonies in vitro. It is widely used to identify and quantify self-renewing mammalian cells derived from in vitro cultures as well as from ex vivo tissue preparations of different origins. Varying research questions and the heterogeneous growth requirements of individual cell model systems led to the development of several assay principles and formats that differ with regard to their conceptual setup, 2D or 3D culture conditions, optional cytotoxic treatments and subsequent mathematical analysis. The protocol presented here is based on the initial clonogenic assay protocol as developed by Puck and Marcus more than 60 years ago. It updates and extends the 2006 Nature Protocols article by Franken et al. It discusses different strategies and principles to analyze clonogenic growth in vitro and presents the clonogenic assay in a modular protocol framework enabling a diversity of formats and measures to optimize determination of clonogenic growth parameters. We put particular focus on the phenomenon of cellular cooperation and consideration of how this can affect the mathematical analysis of survival data. This protocol is applicable to any mammalian cell model system from which single-cell suspensions can be prepared and which contains at least a small fraction of cells with self-renewing capacity in vitro. Depending on the cell system used, the entire procedure takes ~2–10 weeks, with a total hands-on time of <20 h per biological replicate.

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Fig. 1: Overview on the different assay formats and assay principles to determine clonogenic growth.
Fig. 2: Comparison of different assay principles to assess clonal cell growth.
Fig. 3: Schematic overview of the different clonogenic assay procedures.
Fig. 4: Scheme depicting the principle of power regression–based clonogenic survival data analysis.
Fig. 5: An example of the assay optimization procedure for a breast cancer cell line grown in adherent 2D growth.
Fig. 6: Colony morphology displays substantial inter- and intra-cell line heterogeneity.
Fig. 7: High colony density limits the reliability of the colony-counting procedure.
Fig. 8: Intra-assay heterogeneity adds complexity to accurate colony counting.
Fig. 9: Examples of clonogenic survival data analysis.

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Data availability

Source data are provided with this paper. All colony-counting raw data of clonogenic survival experiments in this article (i.e., S-C value pairs of all biological replicates) are provided in the Source Data for Figs. 5 and 9. Some of the clonogenic survival data displayed in Figs. 5 and 9 were taken from ref. 17, as noted in the corresponding figure legends. Schematic graphs in Figs. 2, 4 and 9d were generated from hypothetical datasets. All other data supporting the findings of this study are available within the article and its supplementary information files. Additional information can be provided by the corresponding author upon request.

Code availability

The paper is accompanied by two MS Excel template files for the presented analysis workflows: Supplementary Table 1 for the power regression–based analysis approach and Supplementary Table 2 for PE-based normalization. The R-package CFAcoop is available at https://cran.r-project.org/web/packages/CFAcoop.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft DFG (SFB1321, Project-ID 329628492, P16), the Bundesministerium fuer Bildung und Forschung BMBF (ZiSStrans NUK047A, NUK047C, METABOLiST NUK061C and DKTK) and the International graduate program iTarget (Elitenetzwerk Bayern).

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Author notes

  1. These authors contributed equally: Nikko Brix, Daniel Samaga.

Authors and Affiliations

  1. Department of Radiation Oncology, University Hospital, LMU München, Munich, Germany

    Nikko Brix, Claus Belka, Horst Zitzelsberger & Kirsten Lauber

  2. Research Unit Radiation Cytogenetics, Helmholtz Center Munich, German Research Center for Environmental Health GmbH, Neuherberg, Germany

    Daniel Samaga & Horst Zitzelsberger

  3. Clinical Cooperation Group ‘Personalized Radiotherapy in Head and Neck Cancer’, Helmholtz Center Munich, German Research Center for Environmental Health GmbH, Neuherberg, Germany

    Daniel Samaga, Claus Belka, Horst Zitzelsberger & Kirsten Lauber

  4. German Cancer Consortium (DKTK) partner site, Munich, Germany

    Claus Belka & Kirsten Lauber

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Contributions

N.B. and K.L. conceived and designed the protocol with support from D.S., H.Z. and C.B. N.B. acquired the data. D.S. provided mathematical consultation and developed and programmed the R-package CFAcoop. The MS Excel template files for power regression–based and PE-based survival analysis were generated by N.B. and D.S. N.B. and K.L. wrote the manuscript, and all authors commented on and discussed the final version of the manuscript.

Corresponding author

Correspondence to Kirsten Lauber.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Tatsuya Ohno, Daniel Wahl and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Related links

Key references using this protocol

Brix, N. et al. Radiat. Oncol. 15, 248 (2020): https://doi.org/10.1186/s13014-020-01697-y

Orth, M. et al. Oncogene 37, 52 (2018): https://doi.org/10.1038/onc.2017.304

Hess, J. et al. Cancer Lett. 386, 87 (2017): https://doi.org/10.1016/j.canlet.2016.11.014

This protocol is an extension to: Nat. Protoc. 1, 2315–2319 (2006): https://doi.org/10.1038/nprot.2006.339

Supplementary information

Reporting Summary

Supplementary Table 1

MS Excel template file for power regression–based analysis of clonogenic survival

Supplementary Table 2

MS Excel template file for PE-based analysis of clonogenic survival

Source data

Source Data Fig. 5

All colony-counting raw data

Source Data Fig. 9

All colony-counting raw data

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Brix, N., Samaga, D., Belka, C. et al. Analysis of clonogenic growth in vitro. Nat Protoc 16, 4963–4991 (2021). https://doi.org/10.1038/s41596-021-00615-0

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