( J. Watson SmithKl...)Use of Novel FlashPlate Technology to Measure cAMP Accumulation in Chinese Hamster Ovary Cells Expressing Human -2 Adrenoreceptors

J. Watson
SmithKline Beecham Pharmaceuticals, UK

Introduction

Due to recent technologies in the pharmaceutical industry that have resulted in an increase in the production of potential drug candidates, it has become vital that screening assays are high throughput. FlashPlate technology from PerkinElmer Life Sciences has provided a platform for the adaptation of a variety of assays which can be automated. One such application is monitoring the activity of the enzyme adenylyl cyclase by measuring cAMP accumulation.

Until recently, cAMP has been generated in a biological system (e.g., receptor expressing cells), extracted and then measured by FlashPlate (SMP001) (FlashPlate File #7, J. Watson). However the recent introduction of a novel adenylyl cyclase FlashPlate assay (SMP004, SMP004A) has provided a more efficient method for measuring levels of cAMP from whole cells which eliminates the extraction step (FlashPlate File #1, P. Kasila).

We have investigated the use of this novel FlashPlate technology to measure the activation of adenylyl cyclase in Chinese Hamster Ovary (CHO) cells expressing human -2 adrenoreceptors (h-2 receptors).

Materials

Adenylyl Cyclase Activation FlashPlate Assays (96-well format) were supplied by PerkinElmer Life Sciences. Isoprenaline was obtained from Sigma-Aldrich, and pindolol from RBI chemicals. Hams F-12 media, foetal bovine serum and Geneticin 418 were obtained from Gibco BRL.

Method

CHO cells expressing h-2 receptors were grown to confluence in Hams F-12 media containing 10% foetal bovine serum and 500 μg/ml Geneticin 418, harvested and resuspended in stimulation buffer, containing a phosphodiesterase inhibitor, provided with the FlashPlate kit. The cells were then spun at 200 g for 5 mins. at room temperature, the supernatant removed
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