Cobas fara centrifugal analyser

For F0 and F1 animals, one animal per litter underwent oral glucose tolerance testing (OGTT) (F0 n = 9, F1 n = 5–6). For F2 animals, two animals from each litter were tested (n = 8–14 from 4–7 litters). Following an overnight fast, at 09.00, blood was obtained by tail nicking into EDTA coated micro tubes (Starstedt, Germany) and plasma separated. 2 g/kg of 0.5 g/ml glucose solution (Sigma) was administered by oral gavage. Further blood was collected at 30 and 120 min.
For F0 animals, glucose was measured using a colorimetric kit (Cayman, USA) and for F1 and F2 with a kit (Alpha Laboratories Ltd., UK) adapted for use on a Cobas Fara centrifugal analyser (Roche, UK). Insulin was measured using a Rat Insulin ELISA kit (Mercodia, Sweden,). Fasting plasma triglyceride and cholesterol were determined using kits (Alpha Laboratories Ltd., UK and Olympus Diagnostics Ltd, UK, respectively), adapted for use on a Cobas Fara centrifugal analyser (Roche, UK). Terminal plasma leptin was measured using a Rat Leptin ELISA (Crystal-Chem, IL, USA).

Urine and serum creatinine concentrations were measured using the creatinase reaction, with the exception of the first cohort of the model evaluation studies where Jaffe reaction was used (for the Podo-DTR line 47 given 1ng/g DT). Serum urea was measured using the urease reaction (Alpha Laboratory Ltd, Poole, UK). An immunoturbidimetric assay was developed to measure urinary mouse albumin concentration using a commercial diagnostic Microalbumin Kit (Olympus Diagnostic Ltd, Watford, UK) standardised against purified mouse albumin (Sigma Chemical Co. Poole, UK). All the assays were adapted for use on a Cobas Fara centrifugal analyser (Roche diagnostics Ltd, Welwyn Garden City, UK) according to manufacturer’s instructions.