I've cultured and treated my cells in complete media (containing 10%FBS). Supernatans was collected, centrifuged and stored at -20°C. Now I would like to test ATP release between different conditions. Or will all the ATP have been degraded by the FBS?
I think it's more likely that the ATP would be degraded by storage temperature rather than components of the FBS. I would recommend storing at -80C and reading within about a week.
Dear Julie,
FBS is mainly composed by antibodies, hormones and growth factors. I think that your ATP is not likely to be degraded but, as Jordan said, maybe the best technique to ensure a good ATP conservation is snap-freezing (-80 °C).
Good luck with your research,
Dave
Typically in my experience a single freeze thaw cycle (even if snap frozen in liquid nitrogen) will lead to a loss of ATP by 50% in solution. This is not a problem if all samples are treated the same. The other thing you need to consider, if ATP leaks out of the cell due to loss of membrane integrity, your cells are likely damaged. Therefore it is conceivable that other proteins (i.e. ATPases) will leak out as well (which is the basis of the SDH assay for viability). So while FBS is not the problem, leaky membranes could well be.....
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