SOFT AGAR ASSAY FOR COLONY FORMATION--Protocol 1

Note: All volumes are calculated to cater for four plates per point.

Base Agar:

1. Melt 1% Agar (DNA grade) in microwave, cool to 40C in a waterbath. Warm 2X RPMI + 20% FCS to 40C in waterbath. Allow at least 30 minutes for temperature to equilibrate.

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

3. Add 1.5ml/ 35 m Petri dish, allow to set. The plates can be stored at 4C for up to 1 week.

Top Agar:

1. Melt 0.7% Agar (DNA grade agarose) in microwave, cool to 40C in a waterbath. (It is important not to exceed 40C, otherwise cells will be killed). Also warm 2X RPMI + 20% FCS to the same temperature.

2. Trypsinise cells and count. It is very important to have a positive control line (eg. ras transformed).

3. You require 5,000 cells/plate, therefore you need 20,000/tube. Adjust cell count to 200,000 cells /ml.

4. Add 0.1ml of cell suspension to 10ml yellow capped centrifuge tubes.

5. Label 35mm petri dishes with base agar appropriately (it is a good idea to remove the plates from 4C about 30 minutes prior to plating to allow them to warm up to room temperature).

6. For plating add 3ml 2X RPMI + 10% or 20% FCS and 3ml 0.7% Agar to a tube, mix gently and add 1.5ml to each replicate plate (usually plate out in triplicate). NOTE: Only do one tube at a time so that agar does not set prematurely.

7. Incubate assay at 37C in humidified incubator for 10 - 14 days.

8. Stain plates with 0.5ml of 0.005% Crystal Violet for >1 hour, count colonies using a dissecting microscope.

Protocol 2. Soft Agar Assay for Colony Formation