Miniaturization of the Clonogenic Assay Using Confluence Measurement
AbstractThe 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
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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.
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. International Journal of Molecular Sciences. 2018; 19(3):724.Chicago/Turabian Style
Mayr, Christian; Beyreis, Marlena; Dobias, Heidemarie; Gaisberger, Martin; Pichler, Martin; Ritter, Markus; Jakab, Martin; Neureiter, Daniel; Kiesslich, Tobias. 2018. "Miniaturization of the Clonogenic Assay Using Confluence Measurement." Int. J. Mol. Sci. 19, no. 3: 724.
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