Hi,
I did soft agar assay on MCF-7 cells. I used 0.7% and 0.5% for plate 1 and for plate 2 0.5 and 0.35% of agarose for bottom and upper layers respectively. 5000 cells were seeded in each well. I added 300 ul media weekly to each well for plate 1.
After two weeks, I did not see any colonies in each plate.
Plate 2 was stained after two weeks (because of fungal growth in one well) with 0.5% crystal violet. After an hour of staining, the upper layer was completely destroyed in all wells except in one well and staining was too dark. What could be the reason for upper layer dislodgement?
In plate 1 I did not see any colony even after 4 weeks and I stained this plate after 5 weeks. This time I stained with 0.0025 and 0.005% crystal violet. Although, it was not too dark again it led to upper layer destruction.
In the literature people are getting colonies with MCF-7 cells after two weeks. Anyone who has done soft agar assay with MCF-7 cells, kindly suggest me what could be wrong?.
The protocol of soft agar assay you used seems incorrect. There is a detail protocol of soft agar assay on JOVE (http://www.jove.com/video/51998/the-soft-agar-colony-formation-assay), which could be used as a reference for your experiment. The other important thing is the quality of agarose. The ultra purified agarose is required for doing soft agar colony formation assay.
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