I want to try and determine if the cytokines/growth factors from primary culture prostate cancer associated fibroblasts affect the growth rate of a prostate cancer cell line (LNCaP) through a colony formation assay with conditioned media. Every protocol I have seen uses soft agar CFA but I don't have access to that. How should I go about collecting the CM? How do I store it? Does using CM alter the general steps to performing a colony formation assay? When I did a test run plating 1000 cell/well, with centrifuged and filtered CM for 7 days, the colonies were very small in all my samples and some didn't form colonies at all. Where might I be going wrong? I have attached the improvised protocol I have been using. Any suggestions would be great.
Hi Jessica,
if you use normal cell culture plates, which allow adherence of the LNCaP cells, you are not really performing a colony formation assay, since the colony formation assay is used to analyse anchorage independent growth of tumor cells. Anchorage independent growth is, a lets say, an "old school" marker/property of tumor cells and many cell lines that have been immortalized are derived from single colonies picked from soft agar.
Why don't you have access to low melting agarose (SeaKem® LE-agarose for example) that you would need to perform a soft agar colony formation assay? I know it is not cheap, but if your lab has money for FCS and cell culture media the low melting won't kill the budget, right? An alternative might be the use of plates which don't allow adherence, or Methylcellulose, but I have no experience with these kind of assays.
If you use conditioned media, I would suggest that you mix it with fresh media at least 1:1 to make sure that it is not completely depleted of aminoacids/nutrients etc...
If you need a protocol for a soft agar anchorage independent growth assay aka Colony Formation Assay just let me know!
Best,
Kai
Thank you! I appreciate your response. A protocol would be great. I was able to borrow some agarose from a collaborator's lab so I should be all set.
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