How to Overcome Minor Issues in Anchorage-Independent Assays

Anchorage-independent assays test the ability of cells to grow independent of a solid surface. The assay is used to check the malignant potential of cancer cells. Cancer researchers generally do this experiment for any kind of confirmation of the oncogenic potential of an oncogene or a tumor suppressor in cancer cells. However, we do encounter some issues while doing this experiment. This article lists the minor issues (and solutions) you might face while doing anchorage-independent assays.

Aggravating Agar

The first issue occurs when preparing the agar for the assay. Two problems can occur:

• the agar solidifies before mixing with the media
• the media won’t mix with the agar

To avoid premature solidification of agar, pre-heat a beaker containing distilled water and put the agar-containing flask in that beaker. This prevents the agar from solidifying in the culture hood before you can use it. Also, bring the media to a warm temperature, so that the agar can mix with the media. Keep the required media in a falcon tube in the water bath before mixing with the agar.

Poor Colony Formation in Anchorage-Independent Assays

You may find that colonies do not form properly on the surface of the agar (this makes for a very disappointing day in the lab). High heat from the melted agar might kill the cells when mixing. To avoid this issue, make sure that the top-agar is not too hot before using. Another issue is the proper setting of the bottom agar: keep the bottom agar in a CO₂ incubator at 37°C once it is made.

Too Many Colonies to Count

Sometimes instead of no colonies, you can have the opposite problem—you have too many colonies, and they are difficult to count. This generally happens when you do not do a titration of cells on a soft agar plate. Take the time to do your titration. However, I find that in general, 5,000-10,000 cancer cells on a 6 well plate give a decent amount of colonies appropriate for counting.

Recovering Soft Agar Colonies

Growing back soft agar colonies from the agar is a big hassle. Generally, these colonies do not grow as the agar settles on the plate. To avoid that, use the back of the 200 µl tip and punch the colony lightly to get as little agar as possible. Then, add PBS to the tube containing the colony and centrifuge at 500 x g (slow speeds are preferred, though I have tried different speeds). I’ve used this protocol with NIH3T3 cell colonies cells, but I think it would be work for other cells as well.

Tool for Counting Colonies

Often the question arises: what is the best tool for counting soft agar colonies? You don’t need specialized software to count your colonies. The simplest method I use is the Image J plug-in (Cell counter). Just import the files and use the plug-in to count since colonies are generally big enough to be seen by the naked eye. If you address these issues, you can get good results with anchorage-independent assays.