After primary culture from one tumor sample (of squamous cell carcinoma of cattle ) I found a lot of small round shining apparently nucleated (fine adjusted inverted microscope) cells partly in suspension & partly attached to collagen coated t-25 flasks on the consecutive 3rd & 4th day. Before processing the primary culture the tumor tissue was well detached from vascular tissues. These cells are also increasing in number. When I performed percoll gradient differentiation of cells collected from the flask at different gradient (30%,40,60,80,90%) the cells took the gradient in between 30-40%. Can these be stem cells? Or rather should I ask how to get stem cells from this type of sample? Seeded them in gelatin coated flasks.
Hi Sharadindu,
You can try to confirm if these are cancer stem cells using the AldeFluor assay from Stemcell Technologies. CSC show higher levels of aldehyde dehydrogenase activity, which is measured via flow cytometry in this kit. You can also do cell sorting based on the ALDH signal. If you want to try and culture them, gelatin is a good coating but make sure you add ITS supplement and recombinant IGF-1 (10ng/ml) which will support their growth.
Steve
thank you for your answer I think if these are not RBC or of hematopoietic origin then if I use RBC lysis buffer what may be the consequences? will RBC lysis buffer harm?
I would be careful, the tumor cells are fragile after enzymatic digestion. I re-read your initial question and it is unlikely that the floating cells are tumor cells. Also RBCs can not replicate so if you are seeing the floating cells increase they are not RBCs. The tumor cells should attach. How are you digesting the tumor?
What is the viability of your cells after digestion? Do you see nice epithelial cells attaching in your flasks? The tumor cells will adhere, everything else can be washed out at your first passage. Cold trypsin may not be the the best method for digestion. I use a blend of dispase and collagenasefrom Roche. Look at this website, it is a very good resource
https://www.roche-applied-science.com/webapp/wcs/stores/servlet/CategoryDisplay?catalogId=10001&tab&identifier=Tissue+Dissociation+Overview#tab-0
Let me know if you have other specific questions.
thanks sir. last time I used a proportionate mix of trypsin & stem-pro accutase from life technologies & it worked well.
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