Enzyme Immunoassay for Studying Intracellular Levels of cAMP ( dibutyryl cAMP 2 or 8-bromo-cAMP ch...)

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. Coli⁷ 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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( dibutyryl cAMP 2 or 8-bromo-cAMP ch...)Enzyme Immunoassay for Studying Intracellular Levels of cAMP