Soft Agar Colony Formation Assay Protocol

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for a semi-quantitative evaluation of this capability in response to various treatment conditions.

Short protocol

• Prepare 0.8% base agar layer.
• Prepare 0.7% top agarose solution.
• Harvest cells.
• Count cells and harvest the appropriate number of cells- resuspended harvested cells in top agar.
• Aliquot appropriately on top of the base agar layer (pre-warmed to 37°C).
• Incubate for 10-30 days and feed cells 2 times a week.

Base agar

• Melt 1.2% Agar (molecular biology grade, low melt temp), and cool in a 42°C water bath. Warm 2 x medium + 20% FBS + 2x antibiotics in a 42°C water bath. Allow at least 30 m for the temperature to equilibrate.
• Mix equal volumes of two solutions to give 0.6% agar + 1 x medium + 10% FBS + 1 x antibiotics.
• Add 1.5 mL/35 mm Petri (not tissue culture) dish, allow to set in culture hood. The plates can be stored at 4°C for up to 1 week.

Top agar

• Melt 0.6% Agar (molecular biology grade, low melt temp), and cool in a 42°C water bath (it is important not to exceed 42°C, otherwise cells will be killed). Also, warm 2 x medium + 20% FBS + 2 x antibiotics to the same temperature.
• 200-5000 cells/35 mm plate are required, therefore adjust the number of cells to add into 750 µL of 2 x medium (remember this medium with cells will be diluted 1:2 when mixed with 0.6% agar). For example, for a 1-35 mm dish at 5000 cells/plate, the concentration of cells in 2 x medium will be 6666 cells/mL. Keep at 37°C before plating.
• For each 35 mm dish, mix 750 µL of cells in 2 x medium + 750 µL of 0.6% agar to give you 1.5 mL of 0.3% agar in 1x medium containing 5000 cells, then immediately plate on top of base agar. Assays are usually done in triplicate, so account for 3 wells/condition in calculations; best to make 4 wells' worth for safety.
• Incubate assay at 37°C in humidified incubator from 10 up to 30 days, depending on cell growth. To decrease the risk of contamination, place plates inside a 15 cm tissue culture plate, with an open 35 mm plate of sterile water in the middle for hydration.

Creative Bioarray Relevant Recommendations

• Creative Bioarray offers a soft agar colony formation assay service to support your research. This assay can be used in cell differentiation, cell transformation, and tumorigenesis studies as well as the efficacy evaluation of anti-tumor therapeutics. The procedures of our services include the following.

• Certain cell types are sensitive to the percentage of top agar and the customer needs to determine if 0.3% or 0.4% agar is best suited to the cell type in use.
• Always include a well containing only base and top agar layers, without cells. This will serve as a background control for cell quantification.

Cell Services

Soft Agar Colony Formation Assay Service

For research use only. Not for any other purpose.