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Soft Agar Assay for Colony Formation
Contributor: David Bowtell
University of Melbourne, Australia
 
Procedure
A. Preparation of Base Agar

1. Melt 1% Agar in a microwave and cool to 40°C in a waterbath.

2. Warm 2X RPMI + 20% FCS to 40°C in waterbath. Allow at least 30 minutes for temperature to equilibrate.

3. Mix equal volumes of the two solutions to give 0.5% Agar + 1X RPMI + 10% FCS.

4. Add 1.5 ml of mixture from Step #2 to each 35 mm Petri dish and set aside for 5 min. to allow agar to solidify (see Hint #1).

B. Preparation of Top Agarose

1. Melt 0.7% Agarose in a microwave and cool to 40°C in a waterbath (see Hint #2). Also warm 2X RPMI + 20% FCS to the same temperature.

2. Trypsinize adherent cells to release them and count the number of cells per ml (see Protocol ID#1934). It is very important to have a positive control for colony formation (e.g., A Ras- transformed cell line).

3. This procedure requires 5,000 cells/plate. By using 20,000/tube, there is enough to plate four agar plates from each original tissue culture plate. Adjust the volume so that the cell count = 200,000 cells/ml.

4. Add 0.1 ml of cell suspension to 10 ml tubes.

5. Label the 35 mm base agar dishes appropriately (from Step 3). (See Hint #3.)

6. To plate, add 3 ml of 2X RPMI + 10% or 20% FCS and 3 ml 0.7% Agarose to a tube of cells from Step 4. Mix gently by swirling and add 1.5 ml to each of the three or four replicate plates. Only do one tube at a time so that the agarose does not set prematurely.

7. Incubate plates at 37°C in humidified incubator for 10 to 14 days.

8. Stain plates with 0.5 ml of 0.005% Crystal Violet for more than 1 hour.

9. Count colonies using a dissecting microscope.

 
Solutions
0.005% Crystal Violet
2X RPMI + 20% (v/v) Fetal Calf Serum (FCS)
0.7% (w/v) Agarose (DNA grade)
1% (w/v) Agar (DNA grade)
 
BioReagents and Chemicals
Fetal Calf Serum (FCS)
Agar
Crystal Violet
Agarose
RPMI
 
Protocol Hints
1. These plates can be stored at 4°C for up to 1 week.

2. It is important not to exceed 40°C, otherwise the cells will be killed.