( es. Quench Correction ...)Cerenkov Counting Performance on the TopCountTM Microplate Scintillation and Luminescence Counter

Quench Correction
An experiment was performed to investigate the ability to correct for color quenching. A quench curve containing 151,000 DPM of ³²P per well was prepared in a 96-well OptiPlate using yellow food coloring as the quench agent as described in TopCount Topics, TCA-015.⁵ The total volume of water per well was 200 μL.

Kinase Assays
A kinase assay was performed with the FlexiFilter to test the applicability of the Cerenkov counting technique with TopCount in a routinely performed filter based assay. A protein kinase C (PKC) assay kit (RPN.77, Amersham) was used in the experiment. This assay measures the amount of PKC in a sample by the degree of phosphorylation of a specific peptide with [³²P]-ATP. The assay was performed essentially as described in TopCount Topics, TCA-018.⁴ Serial dilutions of PKC (P-8289, Sigma) were used to generate several levels of specific kinase activity ranging from 125 to 7.8 units of activity per well. Slight modifications were made in this assay compared to the one described in TCA-018. A 25 minute incubation instead of 15 minutes was performed; the stop reagent volume was reduced to 40 μL; and 15 μL instead of 10 μL of activity was spotted onto a precut sheet of phosphocellulose binding paper in a filtration manifold. After the assay was counted on TopCount, the individual sample spots were cut out from the filter and placed with 3 mL of water in glass 7 mL LSC vials and Cerenkov counted on a Packard Tri-Carb 2500TR liquid scintillation analyzer for comparison.

Results and Discussion

96- and 24-Well Microplates and Filter Plates
Table 1 shows the counting efficiency, energy crosstalk, and background CPM for a variety of microplate formats. Note that the crosstalk is not opt
'"/>

Source: