I 'm trying to do the colony formation assay to investigate the long-term activity of my drug. According to the protocols of some papers,MDA-MB-231 cancer cells were seeded at a density of 10000 per well in six-well plates in 2 mL medium. After 24 hr,cells were treated with different concentrations. After washing, fixing and staining, I saw all the colonies have gathered near the walls of the plate (not in the middle for counting) . Does anyone know the reason? This happened due to my fault in the seeding step? or my protocol is wrong?
Dear Ghaffari,
you start with too many cells. I usually start with 500 cells per 2 mL. Please try this protocol .
good luck
There is usually a meniscus effect when plating cells into plates. In our colony formation assays (soft-agar) we first prepare the noble agar/2x media (1:1) plug and after that solidifies, we add the cells (various numbers depending on cell lines) in an agar/2x media+ cells/diluent (2:1:1). When we add the cell layer on, we quickly and vigorously mix in figure eight patterns to spread the agar layer and cells. If the layer is allowed to disperse over the agar plug on its own, the cells congregate near the edges.
Jason
The doubling time of MDA-MB-231 is approx 28-32hr. And as Al Munawir rightly said, the number of cells you used are too high and so you got that effect. 500 or 350 cells per well of 6 well plate should really work well. I used 2 ml for first 48 hr and then replay it with 5 ml and keep them for over 6-7 days to see nice isolated small colonies.
Hope this is helpful
NItesh
In addition to the number of cells plated, it will be helpful if plates or dishes with cell suspensions can be gently moved, not in a circular manner, but in the cross direction (back and forth, right and left), before putting them into the incubator.
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