Pure cancerous cells are needed for the gene quantification after RNA isolation from the cancerous tissue but the contamination is a major problem in biopsy tissues.
If you know the cancer-specific and/or highly expressed marker for the tumor investigated (commonly used are e.g. ErbB-2, cytokeratin7/8, CD326 (EpCAM)) you can also try magnetic separation (MACS, e.g. Miltenyi Biotec). Regards, LZ.
Can you surgically remove the non,cancerous part and then do the collagenase digestion. How big is the tumor specimen, you can do punches
How do you identify non-cancerous cells and cancerous cells within the cancerous tissue. It is quite interesting to know, is there a biochemical or laser dissection can help isolate precise area/region from cancerous tissue.
RKG
Cancer cells are often aneuploid. Based on this principle, there are many protocols available which use flow cytometry to separate cancer cells from normal cells within a dissociated tumor based on ploidy.
If you know the cancer-specific and/or highly expressed marker for the tumor investigated (commonly used are e.g. ErbB-2, cytokeratin7/8, CD326 (EpCAM)) you can also try magnetic separation (MACS, e.g. Miltenyi Biotec). Regards, LZ.
Besides magnetic separation You can also use flow cytometer with cell sorting using specific marker. However, if You have no flow cytometer or magnetic separation is too expenisve You can try to enrich the cell culture in tumor cells using specific conditions of cell culture. Most of the non-tumour cells will die as they are not immortal or will be washed out during passage (e.g., lymphocytes) , however, the big problem are fibroblasts and endothelial stem cells. In book: Culture of Human Tumor Cells (Culture of Specialized Cells) by Roswitha Pfragner and R. Ian Freshney, there are some procedures for enrichment of tumor cell cultures written down and explained. It will not eliminate polluting cells but it could significantly decrease their amount.
Yahoo Schlegel R. et al. from Georgetown Uninversity. They have a very good system for separating cancers cells from normal cells.
Since you don't require live cell, why not consider laser capture dissection to extract pure tumor cells?
We established a co-operation with the pathological department to differentiate between cancer tissue and non-malignant tissue regions. After preparation of tissue samples, of course, we evaluate samples malignity by molecular markers (real-time PCR, Western blotting).
Hello, you can keep passaging for some time so that non cancerous cells will dye or wash out and this will enriched your cancer cells.
According our experience you can use the immunohistochemical marker specific for the particular tumour on the frame slides section 5-7 micrometre - (in accordace with pathology corroboration). You can prepare next pure section that means with nuclei visualisation only on the frame slides. By means of microdissection in the propriate resolution you can than microdissect separate cancer cells. In accordance with aim of your research: with or without immunohistochemistry. Immunohistochemistry can be possibly used for confirmation of tumour cells in comparison to non-stained tissue on the frame slide.
Laser capture will work well on even stained slides. In the absence of this rather expensive technology, the old fashioned micro dissection techniques work.
A lot of thanks for such nice co-operation by the all above researchers & Research gate for providing such nice platform for us,,,,,,,,,,,
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