Many mammalian cell types are anchorage dependent. They need to attach to a solid substratum for them to start to grow. If these cells are forced to grow on a substratum such as a viscous fluid or gel (Example: agar or agarose) where they cannot attach, they fail to grow. However if these cells are treated with certain chemicals and are transformed, they are able to grow in a viscous fluid or gel and become anchorage –independent. This transformed phenotypic change is closely related to the process of in vivo carcino- genesis. For example, many fibroblast cell lines such as BALB/c3T3, NIH-3T3 etc must attach to a solid surface before they can divide. These cell types when suspended in a viscous fluid or gel they fail to grow. However when treated with certain chemicals they transform and gain the ability to grow in viscous fluids such as agarose or agar. This transformation process is very similar to in vivo carcinogenesis. There is a good correlation between in vitro transformation and in vivo carcinogenesis. However the specificity and degree of transformation will vary from system to system being studied. Any positive compounds in this in vitro model are viewed as potential candidates for in vivo carcinogenesis. This is a very reliable and stringent way to screen potential carcinogenic compounds, and carcinogenic inhibitors.
One of the very commonly used assays for colony formation is the soft agar assay. This is an anchorage independent growth assay which is considered as the most stringent for detecting malignant transformation induced by chemical compounds in in vitro model.
Typically the cells of interest are treated with carcinogenic, non -carcinogenic control, or carcinogen inhibitor compounds and are cultured with appropriate positive and negative controls in soft agar medium for 21-28 days. Following the incubation period, some of the cells will form colonies which can be analyzed morphologically by specific staining and quantify by counting number of colonies formed per dish or well.
This experiment can be done in a petri dish, or multiwell plates such as 6 well or 12 well plates. Counting colonies in the petri dish or multiwell plates can be laborious because it is difficult to keep track of the number and size of the colonies. Recently Pioneer Scientific (www.pioneerscientific.com) has introduced unique tools to help with the colony sizing and counting. They have introduced petri dish with numbered grids (Grid Dish) and multiwell plates with grid bottom (Grid plates) Grid Dish are petri dish with 10 mm square numbered grid pattern printed on the bottom side of the dish. These laboratory consumable tools are useful for identifying, counting and keeping track of the colonies in the colony formation assay. Another tool is multiwell plates with grid bottom. Here 12 well or 6 well multiwell plates have 2mm square grid on the bottom. Grids are printed on the bottom side of the well and do not interfere with the cell growth. Multiwell plates with grid bottom are useful in identifying colonies and sizing the colonies because the grids are 2 mm square. By counting the number of cells in a colony of certain size, one can easily size different colony on the grid and calculate the cell number. These set of tools, petri dish with grid bottom, and multiwell plate with grid bottom can make quantifying colonies in the colony formation assay easy to manage.
