Advia centaur insulin assay

The cell line NT-3 was generated from a surgically resected lymph node of a 33-year-old male patient with a well-differentiated neuroendocrine tumor of the pancreas^{24 (link)}. Human insulin content was determined using the ADVIA Centaur Insulin Assay (REF 02230141) and ADVIA Centaur XP analyzer (Siemens Healthcare, Munich, Germany). This assay is standardized to the first human insulin international reference preparation by the World Health Organization (NIBSC code 66/304). Cell culture supernatants of NT-3 cells were analyzed after stimulation with 25 mM Glucose for 60 min in the presence of 20 µM recombinant CEL or CEL-Tn. Cell viability was assessed using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI, USA) after 48 h of 20 µM CEL or CEL-Tn treatment.

The measurement of secreted insulin was performed as previously described. Briefly, ADVIA Centaur Insulin Assay (REF 02230141) and ADVIA Centaur XP analyzer (Siemens Healthcare, Erlangen, Germany) were used. In vitro samples were obtained from supernatants of NT-3 and NT-18 LM cell cultures treated with panobinostat for 24 h and 48 h. Afterwards, stimulation with 500 μmol/L 3-Isobutyl-1-methylxanthin (IBMX, Sigma) for 60 min was initiated. No additional growth factors were added and untreated cells were used as control.

A fasting blood sample was taken by venepuncture for the assessment of leptin, acylated ghrelin, insulin, and glucose. Blood was drawn in EDTA, serum and fluoride collection tubes. Aprotinin (50 μL/mL blood) was immediately added to the EDTA tube for preservation of ghrelin. Plasma and serum obtained were aliquoted and stored at −70 °C until analysis by the Department of Pathology Research & Development at the Leeds Teaching Hospitals NHS Trust (Leeds, UK). All samples analyses were conducted in one batch. Plasma glucose was analysed with the ADVIA Chemistry Glucose Oxidase Concentrated assay (Siemens Healthcare Diagnostics Inc.; UK), serum insulin with the ADVIA Centaur Insulin assay (Siemens Healthcare Diagnostics Inc.; UK), plasma leptin with the Quantikine Human Leptin Immunoassay ELISA kit (R&D Systems Europe Ltd.; UK) and acylated ghrelin with the Spi Bio Acylated Ghrelin Express Enzyme Immunoassay kit (Bertin Pharma; France). The range for the coefficients of variation for intra-assay precision for glucose, insulin, leptin, and acylated ghrelin are 0.2–0.3%, 3.2–4.6%, 3.0–3.3%, and 5.5–10.3%, respectively. Insulin resistance was calculated via the homeostasis model of risk assessment (HOMA-IR) [32 (link)].