( ng pipetting equipment designed for mic...)Cell Proliferation Assays

ng pipetting equipment designed for microplates. After sealing the plate, it can be loaded and counted directly in the TopCount Microplate Scintillation Counter. With up to 12 simultaneously counting detectors, TopCount can cut counting time to 10% of the time required by traditional liquid scintillation counting and decrease radioactive waste.

Experimental

In an experiment to determine the response linearity of TopCount with increasing cell numbers, mouse spleen cells were cultured to a level of approximately 10⁶ cells/ml and stimulated with anti-CD3. The cells were pulsed with approximately 2mCi of [³H]-thymidine for 24 hours, and then increasing volumes of the culture were dispensed into individual microplate wells so that the cell number per well ranged from 10⁵ to 3 X 10⁵. Two plates were prepared, one to be counted on TopCount and another on a discrete vial LSC.

The cells were then harvested onto a Packard UniFilter plate using a MicroMate 196 Cell Harvester. The UniFilter plate is a special microplate, containing a glass fiber membrane and designed to be compatible with the MicroMate 196 Cell Harvester and TopCount. The filters are physically isolated from each other to prevent sample and cocktail migration and to eliminate optical crosstalk between wells. After drying, a reflective backing plate was attached to the bottom of one of the UniFilter plates, and 20 mL MicroScint-O was added to each well. The plate was sealed and counted on TopCount. The individual wells of the other UniFilter plate were removed, placed into 7 ml LSC vials, solubilized, and counted in a conventional LSC.

The count rates increased linearly with cell number (Figure 1). Thus TopCount provides accurate and consistent counting results for cell proliferation assays, across the normal range of cell numbers used in cell proliferation experiments.

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