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Miniaturization of the Clonogenic Assay Using Confluence Measurement.
- Source :
-
International journal of molecular sciences [Int J Mol Sci] 2018 Mar 03; Vol. 19 (3). Date of Electronic Publication: 2018 Mar 03. - Publication Year :
- 2018
-
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.<br />Competing Interests: The authors declare no conflict of interest.
Details
- Language :
- English
- ISSN :
- 1422-0067
- Volume :
- 19
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- International journal of molecular sciences
- Publication Type :
- Academic Journal
- Accession number :
- 29510509
- Full Text :
- https://doi.org/10.3390/ijms19030724