Introduction to clonogenic assays
Clonogenic assay or colony formation assay is an in vitro
cell survival assay that evaluates the ability of single cells to survive 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 and normal cells2.
Live-cell imaging for the analysis of colony formation
Live-cell imaging can address the 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 development, allowing to draw conclusions sooner than when employing a 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 assay results.
In the example below, CHO-K1 cells, treated with a range of doxorubicin concentrations, were imaged for a total period of 7 days using the CytoSMART Omni. The assay was performed in a 24-well plate that was kept inside a standard CO2-incubator at 37C°.
With the CytoSMART colony detection algorithm, clusters of cells (i.e. colonies) formed in an individual well are automatically quantified (Figure 1A) and highlighted in orange (Figure 1B). This allows to monitor and quantify cell proliferation and colony formation throughout the entire incubation period (Figure 2A), without missing any critical time points, as it usually happens when performing a clonogenic assay using a traditional approach. The algorithm also tracks changes in colony size (Figure 2B) and circularity (Figure 2C).
Figure 1 | Analysis of colony-forming ability of untreated CHO-K1 cells over the course of 7 days. (A) The integrated colony detection algorithm automatically detects and calculates the number of colonies per well. (B) Upon zooming in on a particular well, the colony mask overlay highlights individual cell clusters.
Figure 2 | Doxorubicin reduces proliferation and colony formation of CHO-K1 cells in vitro. (A) The outline of a test plate. (B) Survival curve of untreated (CTRL) CHO-K1 cells and cells treated with 0.2 µM, 0.5 µM, 1.0 µM, 2.0 µM, and 5.0 µM doxorubicin, as well as changes in the (C) average colony size and (D) circularity over the course of ~7 days.
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.