• May 29, 2021

Soft Agar Assays for Anchorage Independent Cell Growth

Neoplastic transformation occurs via a series of genetic and epigenetic alterations that yield a cell population that is capable of proliferating independently of both external and internal signals that normally restrain growth. Anchorage-independent growth is one of the hallmarks of cell transformation, which is considered the most accurate and stringent in vitro assay for detecting the malignant transformation of cells.

The soft agar colony formation assay is a common method to monitor anchorage-independent growth, which measures proliferation in a semisolid culture media after 3-4 weeks by manual counting of colonies. This traditional method has been widely published, but manual colony counting can be quite cumbersome, time-consuming, and difficult when testing a large number of samples. Additionally, manual counting of colonies is highly subjective; with varying colony sizes, it’s difficult to determine meaningful results.

Recently advances have been made in the soft agar colony formation assay to increase the speed and accuracy of quantitation. These assays utilize a semisolid agar medium in which colonies are quantified using a fluorometric dye, thereby eliminating manual counting. Because the fluorometric detection method is significantly more sensitive than manual colony counting under a microscope, the assay time is reduced from 3-4 weeks to just 1 week.

It may be of value to further study cell colonies formed in soft agar, in applications such as protein/DNA array analysis or cancer vaccine development. Unfortunately, viable cells cannot be recovered from the traditional soft agar assay. A new advanced colony formation assay system has been developed to allow you to easily recover viable transformed cells for further culturing and testing.

The assays use a special modified soft agar formulation and convey the benefit of the shorter 6-8 day incubation, with the option to either recover the cells or lyse and quantify them using a fluorometric dye. Running replicate samples would allow you to quantify in one well and recover cells from another.

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