I want to separated my tumor cells from fibroblasts and lymphocytes. Then use these cells for global proteomic screening and RNAseq. I use pancreatic tumors and I found it difficult to have high number of viable cells. Someone suggested using the double Ficoll gradient technique to purify my cells. Even when I use a large tumor (1cm^3), I can only get few number of cells. This makes it very difficult to separate the tumor cells after centrifugation. Any recommendations? Also, is it possible to separate Tumor cells from lymphocytes using this technique? Finally, should I put the whole dissociated tissue or filter it with 70um strainer?
Thank you
HI
It is possible to separate the tumor cells with double ficoll. However the yield is low and possibility of getting mixed population. You can prepare the single cells suspension of tumor tissue by enzyme digestion and passing through wire mesh/strainer before ficoll separation. First add 100% ficoll and over it add 70% ficoll (diluted in saline or PBS) and then load the single cell suspension. Centrifuge at 1500 rpm for 20 min at 25 degree celsius. The separation of tumor cells and lymphocytes is also dependent on fatty and fibrous tissue in the tumor sample which always disturb the separation.
Separation of tumor cells using EPCAM- microbeads is also possible. After preparation of single cell suspension, separate EPCAM positive tumor cells immunomagnetically.
Best wishes!
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