I am facing a problem in isolatng adipocytes from rat epididymal fat pads.
By following the Rodbell method, in which fraction are the adipocytes present? At the last step, after centrifugation of collagenase-digested tissue, the stromal vascular cells settle down, and the rest suspension should have the adipocytes.
How do I get adipocytes in a concentrated layer in the final result?
If you wnat just the adipocytes, you don't necessarily need to centrifuge after the isolation. The adipocytes will float into the top of the tube. After some minutes, you can "get" the adipocytes using a pippete (use a tip cut or a properly adipocyte tip) and transfer them to another tube containing PBS (warm). Them they are ready to use.
You can also centrifuge the tubes (just a spin in 1000 rpm), so the adipocytes will be more concentrated at the top.
Patricias' suggestions are straight forward. For further cleaning your adipocyte fraction (and to get rid of collagenase) you may repeat centrifugation in/on PBS or culture medium. Just one question, are you using bacterial (clostridium) collagenase?
Please do not sentrifuge after the Rodbell method since all adipocytes are on the top of the solution.
Regards,
GCK
I guess that's a misunderstanding, (mild) centrifugation may help to concentrate and isolate the top layer of floating adipocytes - as suggested by Patricia already.
Thank you so much for your suggestions. And Sir Dirk Breitkreutz, yes I am using Collagenase type I from Clostridium histolyticum.
I have one more query that the top layer also contains the lipid globules that have come out from the disrupted cells and froth formation is also there in this layer. How do i get rid of it ?
Depending on what you like to do with the adipocytes I have a further suggestion. Though these cells are not very adhesive, you may try plating them on matrix/ECM coated dishes (e.g. collagen type I or IV or BM-laminin) to separate them from lipid vesicles etc. Adipose tissue is surrounded by a basement membrane (BM), so the cells should adhere to respective ECM components.
Thank you so much for your suggestions and help. These are really helpful.
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