I am trying to isolate breast cancer stem cells from breast cancer cell lines through mammosphere formation. I see perfect spheres under microscope but all of them are CD24+/CD44- and dont show cancer stem cell properties.I follow this protocol for isolating:
1-Seeding 20,000 cells/mL in free-serum DMEM/F12 in agar coated flask
2-Adding 20 ng/ml bFGF and 20 ng/ml EGF, 2%B7.
3-Adding bFGF and EGF every two days.
4-Harvesting cells after 7 days.
May you help me why i dont see CD44+ spheres? I am waiting for your response. I hope your helpful response makes me close to my purpose.
Dear Saghar,
ICC is done on whole spheres? Try the cells at the center of the sphere not the periphery.
Mannan
Dear mannan
Thank you so much for your response. If you mean immunocytochemistry, no it is not done. several valuable articles show 90% purity by this protocol without ICC. I use single cells from spheres (center and periphery)
The question is whether these cells are capable of tumorigenesis or not. CD44 often detected in normal stem cells too (example bladder). Try other markers CD133, VEGFR2 etc.
why don't you use FACs sorter. From there you are able to obtain CD24+CD44+.Afterwards, you may validate this population with differentiation assay (adipogenic, osteogenic and etc).
Hello Saghar,
Take into consideration that some cell lines are more enriched for cancer stems cells than others. Growing the cells in 2D and then FACs sorting by stem cell markers should be sufficient. In some cell lines you may get as many as 80% while others you may get just 1%.
Also, the single cell initiating the sphere has stem cell potential but the daughter cells that make up the bulk sphere may be differentiated and again the percentage will be low so you may need to test a very large number of spheres.
Best,
Esmeralda
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