( the reaction was started by the additio...)In Vitro Measurement of the Second Messenger cAMP: RIA vs. FlashPlate

the reaction was started by the addition of viable cells (2 x 10⁵) to LP4 tubes containing forskolin (10 μM) in the presence or absence of 5-HT (10^-10 to 10^-5 M). Basal cAMP levels were determined from cells not treated with either drug. Following a 10 minute incubation at 37C, the reaction was stopped using 30% perchloric acid. cAMP was extracted by addition of a 1:1 mixture of trioctylamine and 1,1,2 trichlorotrifluoroethane, and after vigorous mixing the samples were centrifuged at 3,500 rpm for 15 minutes. The resulting aqueous layer was removed and the level of cAMP determined.

For the RIA method, the samples were dispensed into 1 ml tubes; for the FlashPlate method, the samples were dispensed into a 96-well microplate.

Determination of cAMP Levels using RIA
All reagents were made up as instructed in the RIA Kit (NEK033) manual. The samples were diluted three-fold in a sodium acetate buffer and pipetted manually, in duplicate, into LP4 tubes. The remaining components (i.e., standard cAMP [¹²⁵I] tracer and cAMP antiserum complex) were also added manually. After overnight incubation, binding protein was added to all tubes (except totals). Following mixing, the samples were centrifuged, the supernatant discarded, and the pellets counted on a gamma counter.

Determination of cAMP Levels using FlashPlate Method
The components were made up as per the cAMP [¹²⁵I] FlashPlate Assay (SMP001) manual. The samples were diluted three-fold using a Biomek 1000 Robot (Beckman), then automatically dispensed, in duplicate, into the wells of the cAMP [¹²⁵I] FlashPlates. The standard curve was pipetted manually into the plates, but the cAMP [¹²⁵I] tracer was added by the robot to all wells. After overnight incubation, the FlashPlates were placed onto a Packard TopCount Microplate Scintil
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