Dear All,
At the end of a cell culture experiment, I collected the supernatant and centrifuged it, and did not notice any precipitate. After freezing the supernatant, thawing and centrifuging it, I notice a translucent precipitate. Has anyone experienced that? What could be the nature of this precipitate?
Best regards,
Hello Jeremie Nestor
Repeated freeze-thaw cycles have caused the precipitation to occur. Cell culture supernatant is an attractive source of secreted proteins. It is called spent or conditioned medium that contain growth and other factors the cells have produced for their survival. For instance, whenever one works with primary neurons if one discards all the spent medium the cells will go into apoptosis. The best procedure for neurons and many of the stem cells is to remove only about half the volume of medium at each feed and replace with an equal volume of fresh medium. If you ever have a cell that is growing poorly, try retaining half the conditioned medium and adding an equal volume of fresh medium at each feed until they are growing well, instead of completely changing the medium. This same conditioned medium can also be used to adapt a cell to grow in a new medium. So, you can imagine how precious the spent medium could be.
Repeated freeze-thaw cycles will have a negative influence on proteins present in the cell culture supernatant. It will cause loss of functional properties and conformational changes of protein and can cause the denatured proteins to form insoluble precipitate. It is recommended that the cell culture supernatant be prepared as small-sized aliquots and stored at -20 degree C that will be used up during an experiment. Please avoid freeze-thaw cycles.
Best.
Hi Jeremie Nestor,
I fully agree with Malcolm Nobre . Animal culture media itself is an excellent source of proteins, amino acids, and complex nutrients. Also, spent media (after cell culturing) contains secreted protein and ones that are released from cell lysis.
Repeated cycles of freeze-thawing would result in some precipitation since these proteins undergo misfolding resulting in loss of protein structure and conformation. This is normal, but if you are to work with culture supernatants, then repeated cycles of freeze-thawing should be avoided. Instead, culture supernatants should be aliquoted into multiple vials and used as desired.
Hi, Malcolm Nobre & Jatin Chadha I have a similar question concerning repeated freeze-thawing of spent media. Do metabolite profile such as glucose, ammonium, glutamine, etc gets affected during repeated freeze-thawing of spent media. And also during freeze-thawing of the spent media do microbes present in the environment cause any interference with the metabolites especially ammonium as they can reside on the spent media and consume the nutrients and may produce ammonia content of their own which will give false metabolite results.
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