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Cerenkov Counting Performance on the TopCountTM Microplate Scintillation and Luminescence Counter

des labeled in this way can be used as probes in dot blot assays. The phosphorylation of proteins by kinases is an important cellular control mechanism. In addition, labeling of PCR products with 32P allows easy quantitation of PCR reactions. All of these applications are easily done in a microplate or a microplate format dot blot apparatus. The sample is measured directly in its own medium with little or no preparation for counting.

In this report, the Cerenkov counting performance of the TopCount is documented for linearity, volume effects, efficiency, background and well-to-well crosstalk, as well as the applicability of this technique for liquid and filter/membrane counting. A protein kinase C assay was done to demonstrate the utility of this technique in a common application.

Experimental Methods

The Cerenkov counting mode was used on the TopCount with a factory set region of interest especially suited for highest sensitivity. Where appropriate, samples were also Cerenkov counted on the Packard Tri-Carb 2500TR LSC for comparison to TopCount. The microplates were sealed with TopSeal-A, a self-adhesive sealer. 32P-ATP (Amersham #PB.10204) was the radioisotope used in all experiments. A stock solution containing 49,000 DPM/μL was used for all experiments.

Basic Performance

96- and 24-Well Microplates
Counting efficiency, crosstalk, and background data were acquired for counting in white 96- and 24-well OptiPlates. The 96- and 24-well OptiPlate performance was evaluated using 200 μL and 1.0 mL, respectively, of water per well as the medium. Activity was added to the wells such that a spiked well was surrounded by eight neighboring blank wells containing only water 2 in order to evaluate well-to-well crosstalk. The crosstalk pattern was performed in triplicate for 96-well f
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