This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).
The overall goal of this procedure is to establish patient derived cell cultures from malignant melanomas and isolate putative CD 1 33 positive cancer stem cells. This is accomplished by first preparing primary melanoma single cells from a tumor tissue after depleting erythrocytes from the cell pellet. If necessary, characterize the primary cell culture and deplete the fibroblasts.
The final step is to perform magnetic cell sorting of the CD 1 33 positive and CD 1 33 negative melanoma cells. Ultimately, this optimized max procedure is an excellent method to obtain highly enriched and viable populations of CD 1 33 positive and CD 1 33 negative cells. We first used the technique to validate insco data in personalized medicine where we try to predict drug response of cancer patients and for that no commercially available cell line can be used.
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