Susanne Rasmussen, Anders R. Srensen Novo Nordisk A/S
The free fat cell assay has been used extensively to demonstrate the effect of insulin and insulin analogues on target tissue (1,2). The principle of the assay has been described by Rodbell(3) and Moody et al(4). The assay uses isolated adipocytes from either mice or rats. When stimulated by insulin, the adipocytes incorporate glucose into fat depots, and a suitably labelled glucose added to the medium will result in incorporation of radioactivity into the lipid pool. Extraction of the lipids from the cells has traditionally been performed using a toluene-based scintillator. The phase separation in this system is so effective that direct liquid scintillation counting of the sample is possible without further preparation. The counts originating from the aqueous phase, which still contains a considerable amount of radio labelled glucose, are negligible. Previously, the scintillators that were available for use in the TopCount microplate scintillation and luminescence counter did not have the preferred characteristics for the phase separation, so the toluene scintillator method, with its many undesirable chemical and physical properties, continued to be used. However, the availability of MicroScint-E scintillator from Packard Instrument Company now makes it possible to discontinue the use of toluene-based scintillator solutions, and count samples prepared with MicroScint- E directly in the TopCount.
Animals are killed, and the epididymal fat pads are removed and placed in degradation buffer. Degradation is carried out at 37 C under vigorous shaking for one hour. The cell suspension is filtered in order to remove tissue debris, and the adipocytes are washed twice and resuspended in incubation buffer.
Using the 96-well microplate format, 100 μL of cell suspensio