I seeded some SK-BR-3 spheroids following the protocol in this paper: http://www.ncbi.nlm.nih.gov/pubmed/17982667 but used four different conditions: without contrifugation, 100 g for 3 minutes, 150 g for 5 minutes ant 1000g for 10 minutes. I also used 10 different dilution of cell densities for each plate trying to establish the proper seeding density that will give about 400 micrometer diameter spheroids on day four. While the larger spheroids seem to have better shape with heavier contrifugation. The ones of desired size do not appear to be better, and none of them are of high enough quality.
We need some suggestions to improve the quality of SK-BR-3 spheroids. Should we try higher Matrigel concentration? Is it possible that Matrigel will sediment, so only earlier seeded large ones can get enough Matrigel? Some people also recommended using DMEM/F12 with growth factors and other supplements. Will that work for SK-BR-3 cells, which normally needs McCoy's 5A medium?
I think you need a special medium not regular culture medium you are using for culturing SKBR3. For this kind of assay people always use stem cell medium with growth factors.
1. Use low attachment cell culture plates for sphere assay.
2. Use stem cell culture medium for this assay.
Matrigel might aid in forming more compact spheroids, at least that is our experience. However if you use matrigel be sure to use low attachment cell culture plates. Also judged from the pictures you posted your spheroids does not look to bad.
Hi Kai,
Thanks for the answer. We used ULA round-bottomed plates from Corning. Even with Matrigel, they cannot form compact spheroids. The density is lower compared to MCF7 spheroids. They seem to be composed of many granules, rather than compact homogeneous spheroids like those of MCF7. The shape is quite irregular compared to that of MCF7's. From the photo of the big one I posted, it seems that the center is thinner than the edges, so I think the shape is not even spherical. Maybe it is like the shape of red blood cells.
Dear all: does anyone know the volume/diameter of SKBR3 cell line?
thanks [email protected]
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