Introduction
Several recent studies have revealed that stem cells from various types of self-renewing tissue could be
cultured in suspension. For example, neuronal stem cells grow as neurospheres that do not depend on a
particular substrate. Similarly, mammary stem cells or stem cells from skin can also grow in
suspension using similar culture conditions. These cells differentiate into various cell lineages after
exposure to extracellular matrix molecule (ECM) or serum. Additionally, various cancer stem cells
have been successfully grown under these conditions. These results demonstrate that stem cells can
survive and proliferate in anchorage-independent culture model, and clonogenic activity is a sensitive
indicator of undifferentiated stem cells.
Traditionally, the 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. Standard assays are usually performed in 100-mm or 60 mm dishes, where
cells are allowed to grow inside a semisolid culture media for 3-4 weeks before sizable colonies
appear (> 40 cells/colony). This method is quite cumbersome, time-consuming, and difficult when
testing a large number of samples. Additionally, the manual counting of colonies is highly
subjective; with varying colony sizes, it’s difficult to determine meaningful results.
Cell Biolabs StemTag™ 96-Well Stem Cell Colony Formation Assay does not involve subjective
manual counting of colonies or require a 3-4 week incubation period. Instead cells are incubated only
6-8 days in a proprietary semisolid agar media before being solubilized, lysed and detected by the
patented CyQuant® GR Dye in a fluorescence plate reader or assayed for alkaline phosphatase activity
colorimetrically (Figure 1). Alternatively, viable colony-forming cells (CFCs) can be easily recovered
for further culturing and testing. This format provides a quantitative, high-throughput method to
accurately measure colony formation, while the short incubation time makes it possible to assay
transiently transfected cells.
The StemTag™ 96-Well Stem Cell Colony Formation Assay provides a stringent, anchorageindependent
model for measuring stem cell colony formation, and allows for cell recovery. Each kit
provides sufficient quantities to perform 96 tests in a microtiter plate.