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FREE FAT CELL ASSAY

n 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.

Materials

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.

Buffers:

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.

Results

Extraction of only the lipids is crucial for the interpretation of the insulin action, and the results from MicroScint-E extraction pro
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