Working standards (0.2–25.6 pmol/well) were pipetted into empty wells of the plate. Equal volumes of antisera (20 µl/well), [125I]cAMP (~ 35 000 cpm/well), and Donkey Anti-Rabbit PS SPA Imaging Beads (20 µl, 0.4 mg/well) were added to the microplate wells containing standards and samples. The plates were sealed and incubated at room temperature for 15–20 h, without agitation. The amount of [125I]cAMP bound to the SPA Imaging Beads was determined by imaging on the LEADseeker Multimodality Imaging System for 5 min (2).
The results in Figure 3 show the cAMP produced by CHO cells in response to stimulation by forskolin, an activator of adenylate cyclase. The cells have responded in a dose dependent manner and an increase in cAMP levels can be detected at 1 pmol forskolin, the lowest concentration used.
Figures 4 and 5 show the measurement of cAMP in A431 cells treated with known B2 adrenergic agonist and antagonists. The EC50 values obtained are comparable to those quoted in the literature (3), showing that this imaging assay in 384-well format can be successfully used for high-throughput assays.
Donkey Anti-Rabbit PS SPA Imaging Beads give a good specific signal and large signal window in the cAMP assay. These beads have been utilized in pharmacological experiments and the results correlate very well with other coated SPA beads and non-SPA formats.
Please refer to the SPA imaging bibliography at www.amershambiosciences.com/LEADseeker for a full list of SPA imaging application references.
1. CHIESI, M. et al., Trends in Pharmacol. Sci., 22, (5), 247–254, (2001).
2. PRICE, E. et al., Direct, quantitative, intracellular measu