Anyone knows about isolation and enrichment breast cancer stem cell by culturing MCF-7 in the low attachment surface? In the process to form into a tumorsphere, how often do you add new medium? What are your CD44 CD24 CD133 markers expressions changes?
I normally perform single cell suspension by washing 3x with PBS and adding trispsin for 15 minutes. I pass the cell through a 40um cell strainer to discare aggregates. I always plate single cell suspension in ultralow-attachment 24 well, normally 20.000 cells x single well. The medium is the one used by Dontu ( in Dontu et al., 2003) with all the facotrs. In particular, for each well I usually plate 20.000 cells/500ul medium. Every 3 days I add 100ul of fresh medium with all the factors calculated for the final volume (500ul). I suggest you to add metilcellulose in the medium when you plate the cells at the first time to avoid cells aggregation. After 1 week you can appreciate the tumorspheres formation. As stem cell markers you can use cd44/cd24 or the staining with aldefluor ( you can analyze both with FACS). For the expression changes in stem cells markers beyond cd44/24 and aldefluor, you can analyze the expression of some markers such as slug in tumorspheres versus normal 2d culture.
I suggest you to perform secondary mammospheres instead of only primary mammospheres to discared spheres generated by some progenitors.
I agree with Alessandro's method. In my lab, we use Phorbol 12-myristate 13-acetate (PMA) to stimulate MCF7 cells to obtain ~30% of CD44hi/CD24lo BCSCs. These cells are able to form tumorspheres. Re: the method of measuring BCSCs, you may also want to look at EpCAM (CD326). If you look at epithelial to messenchymal transition, you should see largely increased Vimentin/ Fibroneciton, and decreased E-caherin expression at both protein and mRNA level. Hope it helps.
CD44/24 ratio, CD133, ALDFLUOR, BMI, LGR5, DCLK1, LRIG all these markers mark cancer stem cells but different populations. However, non of them mark all the stem like cells in a given cancer. Colony or single colony selection by mammosphere formation is the good method to isolate possibly all the stem like cells in cancer.
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