I'm trying to do colony formation assays using the breast cancer cell line MM436. So far these methods have failed to get the cells producing colonies:
- 1000 cell seed in 10cm dish, 10% FCS
- 1000 cell seed in 10cm dish, 20% FCS
- 1000 cell seed on top of 60,000 cell (irradiated) feeder layer, 20% FCS
Yet from what I can see in published papers others are using these cells for colony assays, but often under different conditions (e.g. 6-well plate or using an agar layer).
Does anyone have experience using these cells in colony assays? What did you do to get good plating efficiency? I have to use this cell line as they are important for our collaborators' xenograft experiments. Thank you!
I have not worked with the specific cell line mentioned but I have done colony formation with other cell lines. 2000-5000 cells/well of a 6-well plate without an agar layer generally gives good colonies for cell lines I worked with. I did not make any change to the culture conditions so I suggest you to use the routine culture conditions for your cells even during colony formation. Hope this will be useful.
Thank you for your help! However the nature of my experiment is that I need to be able to count the colonies to compare differences with & without drug treatment. If I plated >1000 cells this would get difficult. My results would become meaningless if I plate many thousands of cells to only get a tiny percentage of those cells successfully forming colonies.
I'm going to try using a Matrigel layer to see if this improves things.
I have work on colony formation assay in MCF-7 and MDA-MB-435S cells .I used 1000 cells /well in 6-well plate for 10 days with & without drugs. I found very good result .hope it will help you.
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