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One may need to add a trace element mix such as those described by Ham ef al. , 1976). Many of these trace elements are normally provided as trace contaminants that enter the medium in water or serum. As the medium becomes more defined and the water more pure, these trace elements need to be purposely added to the medium formulation. Sometimes an increased concentration of vitamins can be useful. Vitamin mixtures are commercially available and can be added as such. Some vitamins, such as vitamin E (or a-tocopherol), A (retinol or retinoic acid), or C , are not added to media mixtures because of their instability, but may be important for some cells to survive or function in vitro.
V. Screening Conditioned Medium for Biological Activity It is important to know the composition of the test medium if conditioned medium is to be screened for biological activity. As this is often done at 10-50% conditioned medium, the test medium should minimally use the same bicarbonate buffer concentration as the assay medium and optimally be identical to the assay medium except for changes introduced by the conditioning cells. The fewer the undefined components added to the medium, the easier subsequent purification of any detected activity should be.
It cannot be overemphasized that optimizing medium for one parameter such as growth may lead to a medium that is suboptimal for the expression of a given protein, for response to a given growth factor, or for other physiologic parameters. The medium must therefore be optimized, measuring the parameter for which optimization is desired. X I . Choosing the Optimal Medium: The “Quick and Dirty” Method 1. Obtain and make up medium from several different nutrient mixtures. Be sure and use the appropriate bicarbonate level for the incubator settings (see earlier).