I am trying to isolate cancer stem cells from MCF-7 and MDA MB231 with the help of tumorsphere assay. Is sorting necessary before tumorsphere assay cell seeding or do we sort cells after assay? What is the appropriate number to seed?
First of all it is an enrichment of cancer cells (say in ur case MCF7, MDA-MB-231) to form a cancer stem cells (CSCs) like cells, which is considered as tumorosphere. based on question it is not isolation.
We usually count the cells before assay and then seed them to form sphere. Cell count depends on the planned experiment and culturing condition.
try to follow this protocol I published for melanoma. In my experiences, it worked with MCF-7 too. you can sort if you want test a specific subpopulation, or if you have lots of dead cells that you want to exclude. In regular conditions, with average healthy cells you don't need that.
http://www.bio-protocol.org/e2233 and good luck
I have tried this protocol on Breast cancer stem cells. Yes count your cells before the exp and seed with a constant density in every well. Finally you will have to calculate the mammosphere formation efficiency. refer to the Protocol
Hello everyone,
Counting is necessary but is sorting with specific markers like CD44/CD24 also mandatory before seeding or whether we can seed MCF-7 in non-adherent conditions to form spheres and then do sorting with these markers. I am confused.
Miss Heena,
As I told, it is enrichment process to form CSCs like cells. Once your sphere gets ready you can sort with marker like CD44 and CD24 and make comparison of these marker expression with monolayer culturing of same cell lines.
You can refer our lab paper
The Sphere formation assay is mainly about identifying potential CSCs, so don't sort cells because most of the formed spheres would be of a CSCs origin, after getting your spheres you can now analyze them for other CSCs markers such as (ALDH1A1, Epcam, CD44, CD24, OCT4, SOX2, NANOG)
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