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Int. J. Mol. Sci. 2018, 19(3), 724; https://doi.org/10.3390/ijms19030724

Miniaturization of the Clonogenic Assay Using Confluence Measurement

1
Laboratory for Tumour Biology and Experimental Therapies (TREAT), Institute of Physiology and Pathophysiology, Paracelsus Medical University Salzburg, Strubergasse 22, 5020 Salzburg, Austria
2
Department of Internal Medicine I, Paracelsus Medical University/Salzburger Landeskliniken (SALK), Muellner Hauptstrasse 48, 5020 Salzburg, Austria
3
Laboratory of Functional and Molecular Membrane Physiology (FMMP), Institute of Physiology and Pathophysiology, Paracelsus Medical University Salzburg, Strubergasse 22, 5020 Salzburg, Austria
4
Department for Radon Therapy Research, Ludwig Boltzmann Cluster for Arthritis and Rehabilitation, Institute of Physiology and Pathophysiology, Paracelsus Medical University Salzburg, Strubergasse 22, 5020 Salzburg, Austria
5
Division of Oncology, Department of Internal Medicine, Medical University Graz, Auenbruggerplatz 15, 8036 Graz, Austria
6
Department of Experimental Therapeutics, The UT MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA
7
Gastein Research Institute, Institute of Physiology and Pathophysiology, Paracelsus Medical University Salzburg, Strubergasse 22, 5020 Salzburg, Austria
8
Institute of Pathology, Paracelsus Medical University/Salzburger Landeskliniken (SALK), Muellner Hauptstrasse 48, 5020 Salzburg, Austria
9
Cancer Cluster Salzburg, Institute of Pathology, Paracelsus Medical University/Salzburger Landeskliniken (SALK), Muellner Hauptstrasse 48, 5020 Salzburg, Austria
*
Author to whom correspondence should be addressed.
Received: 11 December 2017 / Revised: 14 February 2018 / Accepted: 1 March 2018 / Published: 3 March 2018
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

The clonogenic assay is a widely used method to study the ability of cells to ‘infinitely’ produce progeny and is, therefore, used as a tool in tumor biology to measure tumor-initiating capacity and stem cell status. However, the standard protocol of using 6-well plates has several disadvantages. By miniaturizing the assay to a 96-well microplate format, as well as by utilizing the confluence detection function of a multimode reader, we here describe a new and modified protocol that allows comprehensive experimental setups and a non-endpoint, label-free semi-automatic analysis. Comparison of bright field images with confluence images demonstrated robust and reproducible detection of clones by the confluence detection function. Moreover, time-resolved non-endpoint confluence measurement of the same well showed that semi-automatic analysis was suitable for determining the mean size and colony number. By treating cells with an inhibitor of clonogenic growth (PTC-209), we show that our modified protocol is suitable for comprehensive (broad concentration range, addition of technical replicates) concentration- and time-resolved analysis of the effect of substances or treatments on clonogenic growth. In summary, this protocol represents a time- and cost-effective alternative to the commonly used 6-well protocol (with endpoint staining) and also provides additional information about the kinetics of clonogenic growth. View Full-Text
Keywords: clonogenic assay; clonogenic growth; 96-well microplate format; microplate reader; confluence detection clonogenic assay; clonogenic growth; 96-well microplate format; microplate reader; confluence detection
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Mayr, C.; Beyreis, M.; Dobias, H.; Gaisberger, M.; Pichler, M.; Ritter, M.; Jakab, M.; Neureiter, D.; Kiesslich, T. Miniaturization of the Clonogenic Assay Using Confluence Measurement. Int. J. Mol. Sci. 2018, 19, 724.

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