I want to start up some simple assays for cAMP levels in cultured cells.
The approach I am going to take is to metabolically label the cells with
[3H]-adenine, and then measure the [3H]-cAMP that is produced from drug
treatments in the assay several hours later.
Ideally, I would like the use cell culture medium that is deficient in
adenine in order to increase the amount of hot adenine that is taken up.
However, when I look over the list of ingredients in D-MEM, I see that
adenine isn't one of them.
Therefore, either the cells are making their own nucleotides, or their
primary source of adenine must be from the FBS that we conventionally add
to the D-MEM.
Does anyone do these kind of assays, and do you typically remove the serum
when you load the cells with hot adenine?
If I can avoid serum-starving the cells, I would prefer to do so.
hines at pharm.med.upenn.edu