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Question about anchorage independent colony formation assay

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#1 gilahari



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Posted 15 August 2010 - 11:58 AM


I am new to this assay and have collected and tried a few different protocols. I am working with various different cancer cell lines.

The problem (?) that I am consistently facing is that cells from the top agarose layer "sink down" through the agarose and adhere to the plastic below. I am seeing this with all cell lines that I am working with. Is this normal? If not, how do I prevent this?

I have tried using the bottom later at 0.5 and 0.7% low melt agarose with a top layer of 0.3%. I am doing my assays in a 6 well cluster plate and using 10000 cells/well.

Any help or advice is much appreciated!


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