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Brix, N.* ; Samaga, D. ; Belka, C. ; Zitzelsberger, H. ; Lauber, K.

Analysis of clonogenic growth in vitro.

Nat. Protoc. 16, 4963-4991 (2021)
Postprint DOI PMC
Open Access Green
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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Publication type Article: Journal article
Document type Review
Corresponding Author
Keywords Hematopoietic Stem-cells; Limiting-dilution; Colony Formation; Hela-cells; Multiple-myeloma; Clonal Growth; Tumor-cells; Radiation; Culture; Assay
ISSN (print) / ISBN 1754-2189
e-ISSN 1750-2799
Journal Nature Protocols
Quellenangaben Volume: 16, Issue: 11, Pages: 4963-4991 Article Number: , Supplement: ,
Publisher Nature Publishing Group
Publishing Place Heidelberger Platz 3, Berlin, 14197, Germany
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) CCG Personalized Radiotherapy in Head and Neck Cancer (KKG-KRT)
Research Unit Radiation Cytogenetics (ZYTO)
Grants International graduate program iTarget (Elitenetzwerk Bayern)
Bundesministerium fuer Bildung und Forschung BMBF
Deutsche Forschungsgemeinschaft DFG