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 suspension per sample is incubated with 10 μL glucose solution and 10 μL insulin for two hours at 37 C using gentle shaking. The incubation is stopped by adding 150 μL of MicroScint-E scintillator, and counted.
Collagenase is obtained from Wellington, radio labelled D-3-3H-glucose is from Amersham Pharmacia Biotech, and human serum albumin is from Behring Diagnostics GmbH. MicroScint-E is obtained from Packard Instrument Company. All other chemicals are analytical grade or higher.
Weltmans Reagent: 66.2 g CaCl22 H2O, Water ad 1000 mL.
Krebs Stock: 32.26 g NaCl, 1.77 g KCl, 0.811 g KH2PO4, 1.469 g MgSO47H2O, 28 mL Weltmans Reagent, water ad 1000 mL.
10% Human Serum Albumin (HSA): 52.5 g HSA is dissolved in 250 mL water. Add 26.25 g activated charcoal and let it stand for at least 4 hours (room temperature). Shake occasionally. Filter mixture to remove charcoal and dialyse to remove ions. Dilute to 500 mL.
Hepes pH 7.9: 11.92 g Hepes is dissolved in 50 mL water. Adjust pH to 7.9 using 1N NaOH. Dilute to 100 mL. Hepes Buffer: 40 mL Krebs, 50 mL 10% HSA, 10 mL 0.5M Hepes pH 7.9 Water ad 200 mL.
Incubation Buffer: 100 mL Krebs, 50 mL 10% HSA, 25 mL Hepes pH 7.9, water ad 500 mL.
Degradation Buffer: 4 mg collagenase, 2 mg glucose, 4 mL 10 % HSA, 2 mL Krebs, 0.5 mL Hepes pH 7.9, 3.5 mL water.
Glucose Solution: 12.5 μL D(3H3) glucose 1 mg glucose 1 mL Hepes buffer.
Extraction of only the lipids is crucial for the interpretation of the insulin action, and the results from MicroScint-E extraction proved to be comparable to those from using the toluene scintillator extraction system. Using the same batch of cells for comparing the two extraction systems gave ED50 of 103 10 pM for toluene scintillator and 97 7 pM for MicroScint-E. The full dose response curves are shown in Figure 1.
The MicroScint-E scintillator from Packard Instrument Company provides the characteristics necessary to extract and count free fat cell assay samples in 96-well microplates in the TopCount without physically removing the aqueous phase. This heterogeneous counting method simplifies the assay, and enables rapid preparation and multiple-detector counting of such assays. MicroScint-E also eliminates the hazardous properties of toluene scintillator system, which was formerly the extractant of choice.
1) Brange J, Ribel U, Hansen JF, Havelund S, Melberg SG, Norris F, Norris K, Snel L, Srensen AR, Voigt HO (1988). Nature, 333, 679-682.
2) Vlund Aa, Brange J, Drejer K, Jensen I, Markussen J, Ribel U, Srensen AR, Schlichtkrull J. Diabetic Medicine 1991; 8: 839-847.
3) Martin Rodbell. (1964). The Journal of Biological Chemistry. 239(2).375-380.
4) A. J. Moody, M. A. Stan, M. Stan. (1974), Horm.Metab.Res.6.12-16.