We often use soft agar colony formation assay to test cancer cells' Anchorage-independent growth as a hallmark of carcinogenesis, the assay described by "Stanley Borowicz, et. al.".
Yet, I can find no detailed evidence of how to use such assay for cancer cells' transformation response to specific treatments.
Do you know of the best timing to include the treatment for such experiment?
Can I include the treatment in the upper agar/cells layer mix from the beginning?
OR
Should I keep fortifying the surface with treatment during the whole 20+ days duration of the experiment?
OR
Should pre-treatment of cells can be the best option, prior starting the soft agar colony formation assay?
For technical ease, and to ensure that you treatment reaches the cells properly, I would suggest you start by doing a pre-treatment prior to embedding in soft agar. Good luck!
As per my experience, the best way to treat the cells with drug/compound while plating and replace every 48-72 hours. Stop the treatment at least 48 hours before the termination (2-3wks depending on cell type and colony). You can follow the below protocol-
Article Utilization of the Soft Agar Colony Formation Assay to Ident...
Best,
Dear Husam,
As you know this assays measure the capacity of the cell to resist the anoikis ( apoptosis induced by lack of correct cell/ECM attachment ). In my experience the best experiment design is pre-treat yours cells with your drug , for the time that you choose, and after that wash the drug and perform the soft agar assay. In this way you can measure how your treatment affect the capacity of your cell to resist the anoikis.
Good luck
Cynthia
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