Hi all! I have recently harvested the conditioned medium from different colorectal cancer cell lines. But some of the flasks wouldn't freeze at -20, and hardly start to get solid at -80. What may be the reason for that?
Hi Valeriia.
Mucin components secreted from colorectal cancer cells tend to form gel absorbing lots of water from the conditioned medium, and serum proteins also help the rigidity of the generated gel form. The different amounts of mucins from different cell lines you used reflect the differences in the extents of freezing.
Regards.
Hello Valeriia.
If you would like to collect and test biological activities of the conditioned medium, the collected medium should be spun down at ~5,000-10,000xg at 4C. Save the upper part of the fluid carefully, followed by milipore through 0.22um pore size. Make aliquots in smaller tubes for storage at -70C. This will eliminate all the thick part of secreted materials, cell debris and invisible membranous vesicular structures (i.e., exosomes).
When you use 10, 20, 30 and 40% ....of the fresh culture medium, replace 5% FBS and glucose (same concentration as in fresh medium) of the added amount of the conditioned medium.
The conditioned medium is usually recovered at 48h for maximum effects of the activities. Thus, the conditioned medium would have lower pH, leading to lower pH of fresh culture medium, often masking the activities of the conditioned medium.
Needs attention to evaluate its activities.
Best wishes.
I would agree with Sang Ho Lee to a point. But add following modifications. "Freeze / thaw" conditioned x 3 at -20C (fracture any existing cells), spin at 100,000 x g at 4C (pellet debris, other items in suspension), remove supernatant, and sterilize through a 0.10 micron pore size. Aliquot, but store at -80C (will suspend degradation, any higher temperature and there will be a definite shelf-life). If you use serum in your cultures, heat inactivate it first, otherwise inductive factors in your conditioned medium will be competing with those factors in the serum.
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