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Enzyme Immunoassay for Studying Intracellular Levels of cAMP

dibutyryl cAMP2 or 8-bromo-cAMP, cholera toxin (CT),2 3-isobutyl-1-methylxanthine (IBMX)3 and forskolin.4 AC toxin has been shown to elevate intracellular cAMP in cultured cells5 and can be used as an alternative to agonists that increase cAMP levels. AC toxin has an apparent molecular weight of 200 to 216 kDa by SDS-PAGE.6 Stratagenes AC toxin has been purified from E. Coli 7 and is supplied as lyophilized powder sealed under vacuum. When reconstituted in 50 l distilled water, each vial contains 50 g of AC toxin in 20 mM HEPES, pH 7.5 and 8 M urea.

AC toxin activity was determined by quantitation of intracellular cAMP accumulation in Jurkat8 cells using Stratagenes EIA cAMP Kit. Jurkat cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 50 units/ml penicillin and 50 mg/ml streptomycin in a humidified, 370C, 5% CO2 incubator. Various concentrations of AC toxin were added to the cells (1 x 106/ml), and intracellular cAMP levels were quantified.

figure 3

AC toxin elevates intracellular cAMP levels in Jurkat cells (figure 3). cAMP levels were elevated by as little as 0.5 g/ml of AC toxin, although 2.0 g/ml seems to be the optimal concentration for elevating values of cAMP. A 5-fold increase in the AC toxin concentration to 10 g/ml resulted in decreased intracellular cAMP levels, suggesting that cAMP production levels off at high concentrations of AC toxin.9 Stratagene recommends that researchers vary levels of AC toxin within this range (0.5 to 10 g/ml) to ensure maximal stimulation in the cell line of in
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