Hello there,
I am working to isolate breast cancer stem cells from MDA-MB-231 using CD44+/CD24low positive selection on magnetic separation (Miltenyi Biotech column). Has anybody used this kit for solid tumor cells and not hematopoietic cells that the protocol is based on? I reach a good enrichment but I end with very low number of cells to work with. Any suggestion is helpful.
if you get the very low number of stem cells afterwards - you did a very good job and the protocol worked very well as there is not supposed to be really many stem cells in the culture ( only about 1-4% out of all cells). To get more stem cells - you just need to use more of cells at the beginning. Trypsinize cells well and check if there is no conglomerates. You might consider stimulation of stem cell proliferation before hand to increase stem cell population.
Yes the yield of stem cells that you will get will be very low, as it is a magnetic based separation, and the amount of starting material required is very high, which in your case is a fresh tumor tissue.You will need to process the tissue carefully and make sure that you have got a homogeneous cell suspension and also ensure the viability of the cells.
I have used these kits for isolation of spermatogonial stem cells.
As far as i know, almost all MDA-MB-231 cells are CD44+/CD24low or negative. I would suggest to perform flow cytometry before and after purification. A caution: If you are detaching cells with extensive trypsinization, then the cells may loose certain surface markers (potentially CD44 as well).
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