Introduction to clonogenic assay
Clonogenic assay or colony formation assay is an in vitro cell survival assay that evaluates the ability of single cells to survive a treatment and reproduce to form colonies1.Clonogenic assays were initially developed to study the effects of radiation on mammalian cells. As of late, they have been widely used to test the efficacy of anti-angiogenic and cytotoxic agents, chemotherapy and cytokine-based drugs on both neoplastic or normal cells2.
Traditional protocol for clonogenic assay
A traditional clonogenic protocol involves seeding cells at low density and examining plates after 1 – 3 weeks for the formation of colonies (Figure 1). A single colony (i.e. clone) is made up of direct progeny of a single cell that has undergone “unlimited” division1,2. To visualize colonies, cells need to be fixed with 6.0% glutaraldehyde and stained with 0.5% crystal violet. Subsequently, the colonies can be counted with a light microscope and a survival curve is plotted1. A cell survival curve represents a relationship between the cytotoxic agent and the proportion of cells that survive the treatment2.
Figure 1. A schematic representation of a traditional clonogenic assay setup. The figure is reused from “Clonogenic assay: what, why and how” by Dr. Jenna Bleloch (CytoSMART Resources).
Although a clonogenic assay is a relatively straightforward procedure, there is a number of issues to consider when performing this assay using a traditional approach:
- Clonogenic assay involves only the endpoint analysis of the sample, thus no information can be retrieved regarding the progression of the experiment and the exact point of time when the treatment starts to affect the cells
- The assay is labor-intensive and time-consuming due to the prolonged duration of the experiment, multi-step experimental setup, and tedious counting of colonies
- Crystal violet staining is toxic to the cells, so once the staining is applied, the cellular sample cannot be used for further analysis/processing
- Manual colony counting and result interpretation can be subjective, resulting in user-to-user variation
- When following a traditional clonogenic assay protocol, it is only advisable to use culture dishes and 6-well plates to avoid merging of colonies, which lowers experimental throughput
- Certain treatments (e.g. si-RNA- or Cas9-based) might be too expensive to include when using a 6-well format, as larger volumes of media/drugs are needed3
Live-cell imaging for the analysis of colony formation
Live-cell imaging can address the aforementioned drawbacks of a traditional clonogenic protocol. Firstly, live-cell imaging allows to monitor colony growth over long periods of time not limiting data acquisition to a singular time point. This allows to gather more information about the survival of cells following a specific treatment, as well as observe cell division and colony formation in real-time. With continuous imaging, colony formation can be detected at a much earlier stage of the development, allowing to draw conclusions sooner than when employing a standard/traditional approach. The absence of endpoint fixation or staining steps further reduces the time and materials it takes to conduct the clonogenic assay. In addition, the integrated image analysis software automatically detects newly formed colonies and evaluates their size and circularity. This not only accelerates data analysis, but also reduces bias and subjectivity in the interpretation of results. The key features of traditional and live-cell imaging approaches are summarized in Table 1.
Table 1. Comparison of traditional and live-cell imaging approaches for clonogenic assay.
1. Franken, N. A. P., Rodermond, H. M., Stap, J., Haveman, J. & van Bree, C. Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315–2319 (2006).
2. Munshi, A., Hobbs, M. & Meyn, R. E. Clonogenic Cell Survival Assay BT - Chemosensitivity: Volume 1 In Vitro Assays. in (ed. Blumenthal, R. D.) 21–28 (Humana Press, 2005). doi:10.1385/1-59259-869-2:021.
3. Mayr, C. et al. Miniaturization of the Clonogenic Assay Using Confluence Measurement. Int. J. Mol. Sci. 19, 724 (2018).