Cobas b101 poc system

Glycated hemoglobin A1c (HbA1c) was measured using a Cobas B101 POC system (Roche, Basel, Switzerland). Briefly, 2 μL heparinized blood was placed into the Cobas cartridge with TRIS buffer and sodium lauryl sulphate (SLS) buffer to form a SLS-Hb complex. Detection at 525 nm provided a measure of total Hb and the HbA1c fraction was measured via a latex agglutination immuno-turbimetric reaction at 625 nm. % HbA1c value was calculated using a ratio of the concentration of HbA1c to total Hb (n = 3/group).

Fortnightly and endpoint collection of blood from diabetic mice was used for evaluation of hyperglycemia (≥26 mM), and hence onset of T1D. Saphenous vein bleeds were conducted fortnightly following STZ or citrate vehicle administration and glucose was measured using a glucometer (Accu-check Advantage; Roche, Switzerland). At endpoint, whole blood collected via cardiac puncture was assessed for glycated hemoglobin (HbA1c) using a Cobas b101 POC system (Roche, Basel, Switzerland) and differential blood cell count using a HEMAVET^® Hematology Analyzer 950 (Drew Scientific, Miami Lakes, FL, United States). Plasma TNFα and IL-1β were measured using mouse-specific colorimetric assay kits¹ (Melbourne, Australia) in a subset of animals from each group. Water and food consumption and urine output were measured using metabolic cages over a 24 h period (this was not measured for mice in the 2 week group and only a subset in the 16 week group).

At study end, animals received an endpoint dose of ketamine/xylazine (85/8.5 mg/kg i.p.). Blood samples were collected by cardiac puncture into heparinised tubes and centrifuged at 3,000 g for 15 min to obtain separate cell and plasma fractions. Plasma samples were collected for subsequent analysis. The heart and other organs including gastrocnemius, tibia, kidney and liver, were collected. The heart was dissected into the atria, right ventricle and left ventricle. A section of the LV was collected for histology and a section was snap-frozen in liquid nitrogen and stored at −80°C for RNA extraction and RTPCR. Glycated haemoglobin (HbA1c) was measured at study end using the Cobas b 101 POC system (Roche). Circulating plasma insulin concentrations at endpoint were measured by mouse-specific insulin ELISA kit (cat no. 80-INSMSU-E01, ALPCO), as per manufacturer’s instructions.