Dear all,
I would like to isolate monocytes from buffy coats and further cultivate and differentiate them in the inactivated plasma (supplemented with M-CSF) obtained during centrifugation of buffy coat on Ficol gradient. Is it possible to use this plasma instead of human serum? Should I get rid of fibrin?
Dear Jakub,
I'm afraid not. First because usually you have to dilute the blood with medium or PBS before you can layer it on top of the Ficol. Second the Ficol is extremely toxic and the trace present in this plasma will be toxic to the cells during culture. My advice is to sample 10 ml of blood in a separate tube to prepare autologous inactivated serum and use it at 10%.
Hello Jakub
I agree with Mireya. You should not use this plasma mixture with Ficol. Ficol is toxic to cells. In addition, the buffy coat plasma is not suitable because it has a high concentration of residual metabolism (few volume of plasma versus high cell concentration).
Best regards
You really need to save the serum/plasma prior to the ficol step. You can add some Ca + Mg after removing the buffy coat to produce the serum, but don't heat-inactivate it. Good luck.
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