Colony Forming Assay

Colony forming assay, also called the clonogenic assay, is an important technique in cellular biology. The method is used to observe the reproductive viability of cells, providing insight into its ability to survive, proliferate and form colonies. The applications of the assay are broad, including cancer studies, toxicology, and genetic research. Understanding the technique is important for researchers to interpret the cellular response to different treatments.

Colony Forming Assay

---

Colony forming assay measures the ability of a single adherent cell to grow into a visible colony. When a single viable cell is plated under optimal conditions, it divides and forms a colony. This ability is a key indicator of the cell's capacity to survive long-term and proliferate. The technique is widely used in microbiological cultures and while the applications vary, the assay is particularly useful when testing the cell's response to treatments, such as radiation, chemotherapy, and other drugs. Colony formation assay was first used to examine how radiation affects cancer cell survival. It remains the gold standard for studying cell reproductive death following radiation exposure until today. Apart from cancer research, the assay is implemented in toxicology and genetic studies.

Materials and Method

---

Materials

• Adherent cells with appropriate culture medium
• Crystal violet stain or methylene blue
• Paraformaldehyde
• Phosphate-buffer saline (PBS)
• Trypsin
• Bright-field microscope
• Centrifuge
• Centrifuge tubes
• Colony counting software
• Incubator
• Laminar flow hood
• Micropipettes
• Tissue culture plates

Method

Harvesting and Plating the Cells

1. Harvest the cells by removing the media from the flask. Add 2 ml of trypsin and incubate the flasks for 1 minute so the cells detach. Add 2 ml of media to the flask to neutralize the trypsin. Pellet the cells by centrifugation at 1000 rpm for 5 minutes. Resuspend the cells at 200 cells/ml.
2. Add 1 ml of cell suspension to each well of the 6-well or 12-well plate. The number of cells can be adjusted as needed.
3. Incubate the cells for a specific time at 37°C.
4. Check the formation of the cells with a microscope. Once the colonies are at the required size, stain them and count them.

Fixing and Staining the Cells

1. Gently remove all the medium from the plates. Add paraformaldehyde to cover the cells and incubate at room temperature for 20 minutes.
2. Remove the paraformaldehyde and wash the cells with PBS.
3. Add the staining solution to cover the cells. Incubate the plates for 5 minutes at room temperature.
4. Wash the cells with PBS to remove excess dye.
5. Invert the plates on tissue paper to dry overnight.
6. View the plates under a microscope and count the colonies.

Counting and Data Analysis

Once stained, counting and analysing the number of colonies is an essential step to draw conclusions from the assay. Counting the stained colonies can either be performed manually using a stereomicroscope or with an automatic colony counter. While counting the colonies manually ensures precise identification, automated counting offers higher throughput and minimizes human error.

The first step in data analyses is calculating the plating efficiency. This represents the percentage of seeded cells that have successfully formed a colony. It provides insight into the health and viability of the cells. For more detailed analysis, you can study the colony size and number. This reveals the effect of the treatment on the colony formation, where a decrease in either may indicate the cytotoxic effects. To strengthen the conclusion, it is recommended to perform statistical analysis such as ANOVA or t-test, which compare results across different experimental conditions.

Key Considerations for Colony Forming Assay

---

Counting the number of cells before seeding is a crucial step to ensure consistent seeding densities in all experimental replicates as it directly impacts the reliability of the assay. This can be carried out by using haemocytometer or an automated cell counter. The variation in seeding density has a direct impact on the number of formed colonies, impacting the data interpretation. When seeding the cells, it is necessary to ensure even distribution. This minimizes cell clustering which can lead to inaccurate colony counts. Even distribution can be ensured by gently swirling the plate after adding the cell suspension. Equally important factor to consider is the selection of culture plates. As this directly impacts the results, it is important to choose plates that support colony growth while also allowing clear visualisation and quantification.

Applications in Cancer Research and Beyond

---

Colony forming assay is used in cancer research to evaluate the effectiveness of chemotherapeutic agents. The drug's cytotoxic potential is measured by the reduction in the numbers of colonies formed after the treatment. It is a critical step in developing targeted therapies, as the method allows researchers to study how novel anticancer compounds impact cancer cell viability.

Colony forming assay is used in toxicology and environmental studies to assess the long-term cytotoxic effects of chemicals and environmental substances. The potential health risk is evaluated based on how the substance affects colony formation.

In genetic studies, colony forming assay helps identify gene functions that are directly related to cell growth and cycle regulations. Researchers can explore how specific gene changes affect the cell's behaviour by comparing wild-type genes with modified genes.

Advantages of Colony Forming Assay

---

• Measures long-term cell survival and reproductive viability: Colony forming assay provides insight into how a single cell proliferates over time.
• Enables qualitative and quantitative analysis: As colonies are stained, they are easily visualised. This provides a clear and direct representation of the outcome. Not only can you evaluate the size of the colonies, you can also count them.
• Applicable for different research fields
• Provides insight into cellular mechanisms: The assay helps evaluate the mechanisms of cellular survival, death and resistance by identifying the cells that have the capacity to proliferate after treatment.

Limitations of Colony Forming Assay

---

• Labour-intensive and low throughput: Counting the colonies manually is time-consuming and restricts scalability, specifically when counting in formats bigger than 12- or 24-well plates.
• Subjectivity in scoring: Although the standard definition of a colony is a group of 50 or more cells, subjectivity can influence how the colonies are counted. This can lead to variability in results between researchers.

Learn More

---