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Utilization of Donkey Anti-Rabbit PS SPA Imaging Beads in the quantitative measurement of cAMP in cells

ultured cells utilizing Donkey Anti-Rabbit PS SPA Imaging Beads to generate signal in a simple, homogeneous format (Fig 2).


Materials
Products used
LEADseeker Multimodality Imaging System 18-1140-71

Donkey Anti-Rabbit PS SPA Imaging Beads RPNQ0299


Other material required
Assay buffer

cAMP standard

Adenosine 3',5'-cyclic phosphoric acid 2'-0-succinyl-3-[125I] iodotyrosine methyl ester, 147.5 kBq, 4 µCi, lyophilized.

Rabbit anti-succinyl cAMP serum, lyophilized.

Lysis reagent 1

Lysis reagent 2

The above reagents are available in the cAMP – [125I] Direct Biotrak™ SPA Screening Assay (RPA 559).


Method
Chinese hamster ovary (CHO) or A431 cells were seeded into 384-well cluster plates suitable for both cell culture and imaging. Cells were stimulated (20 min 37 °C, 5% CO2, 95% humidity) either with forskolin (1–1000 µM) (Fig 3), one of the β-adrenoreceptor agonists: isoproterenol, epinephrine, salbutamol, or noradrenaline (1.25–1000 nM) (Fig 4), or pre-incubated for 30 min with the non-selective adrenoreceptor antagonist propranolol (0.001–1000 nM), before stimulation with isoproterenol (1000 nM) (Fig 5) (1).


A two-stage method was applied, where the cells were cultured and lyzed in a separate vessel. The culture supernatant was gently aspirated and the cells lyzed with 0.25% (w/v) dodecyltrimethyl ammonium bromide. An aliquot of lysate was transferred to a second plate for assay.
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