Abstract
Clonogenic assays are powerful tools for testing cell reproductive death after biological damage caused by, for example, ionizing radiation. Traditionally, the methods require a cumbersome, slow and eye-straining manual counting of viable colonies under a microscope. To speed up the counting process and minimize those issues related to the subjective decisions of the scoring personnel, we developed a semi-automated, image-based cell colony counting setup, named CoCoNut (Colony Counter developed by the Nutech department at the Technical University of Denmark). It consists in an ImageJ macro and a photographic 3D-printed light-box, conceived and demonstrated to work together for Crystal Violet-stained colonies. Careful attention was given to the image acquisition process, which allows background removal (i.e. any unwanted element in the picture) in a minimally invasive manner. This is mainly achieved by optimal lighting conditions in the light-box and dividing the image of a flask that contains viable colonies by the picture of an empty flask. In this way, CoCoNut avoids using aggressive background removal filters that usually lead to suboptimal colony count recovery. The full method was tested with V79 and HeLa cell survival samples. Results were compared to other freely available tools. CoCoNut proved able to successfully distinguish between single and merged colonies and to identify colonies bordering on flask edges. CoCoNut software calibration is fast; it requires the adjustment of a single parameter that is the smallest colony area to be counted. The employment of a single parameter reduces the risk of subjectivity, providing a robust and user-friendly tool, whose results can be easily compared over time and among different bio-laboratories. The method is inexpensive and easy to obtain. Among its advantages, we highlight the possibility of combining the macro with a perfectly reproducible 3D-printed light-box. The CoCoNut software and the 3D-printer files are provided as supporting information (S1 CoCoNut Files).
Highlights
The clonogenic assay is a well-established method for measuring cell response to damaging agents, for example ionizing radiation
The tool was positioned inside the light-box; pictures of the target have been realized by using the interactive 3D surface plot ImageJ plugin
Results show a uniform distribution of light, with a reasonable signal drop from side c toward side a (Fig 6), which can be explained by the absence of a LED strip on the side of the door
Summary
The clonogenic assay is a well-established method for measuring cell response to damaging agents, for example ionizing radiation. The response is a relationship between the quantity of the agent (the absorbed dose) and the fraction of cells that retains the ability to reproduce and form colonies within a given amount of time. Colonies with more than 50 cells are identified as viable and counted [1]. Adoption normally makes viable colonies conveniently detectable to the naked eye, depending on the cell line. It is always necessary to work with a large number of samples, which turns the colony counting process into a time-consuming and eye-straining task. We present a new Colony Counter, developed by the Center for Nuclear Technologies (Nutech) at the Technical University of Denmark (DTU). The new tool is named CoCoNut (Colony Counter developed by the Nutech department at DTU)
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