Au400 analyzer

To evaluate liver and renal function after CPB, clinical chemistry was carried out. Renal function was evaluated by serum creatinine and blood urea nitrogen (BUN). Liver function was analyzed using alanine transaminase (ALT) and aspartate transaminase (AST). All measurements were done on an AU 400 Olympus analyzer according to the manufacturer´s instruction.

Alanine transaminase (ALT) and aspartate transaminase (AST) were measured using an AU 400 Olympus Analyzer (Olympus, Tokyo, Japan) as previously described [22 (link)]. TNF-α, IL-6, MIP-1α, and IFN-γ were measured using a cytometric bead-based (CBA) immunoassay (BD) according to the manufacturer’s instructions. Sample acquisition was performed using a FACS Canto II with FACS Diva software, and data analysis was conducted using FCAP array v3 software (Soft Flow Inc, Pecs, Hungary).

Lactatdehydrogenase (LDH), Alanine transaminase (ALT) and aspartate transaminase (AST) were measured by using an AU 400 Olympus Analyzer (Olympus, Tokyo, Japan) as previously described^{24 (link)}.

For the BrdU incorporation assay 2000 cells per well were seeded in a 96-well culture plate. 24 hours later, BrdU labeling solution was added for 4 hours. BrdU incorporation into DNA was quantified with the Cell Proliferation ELISA kit (Roche). Blood was collected from posterior vena cava. It was left to clot for 1–2 h at room temperature and centrifuge at 1000G to obtain blood serum, which was immediately frozen at −80°C for posterior analysis. Cross-linked N-telopeptides of type I collagen (NTX) in mouse serum were measured by ELISA kits (Cusabio Biotec, CO). Alkaline Phosphatase (ALP) levels were determined following the recommendations of the International Federation of Clinical Chemistry. For calcium and phosphate concentration, the arsenazo III and molibdate methods were applied respectively. ALP, calcium and phosphate levels were analyzed at the Clinical Biochemistry Service of the Faculty of Veterinary Medicine (Universitat Autònoma de Barcelona) using an Olympus AU400 analyzer.

Venous blood samples were collected after an overnight fast for determination of serum insulin, glucose, triglycerides, and total and high-density lipoprotein cholesterol (HDL-C). Low-density lipoprotein cholesterol (LDL-C) concentration was calculated using the Friedewald equation [31 (link)]. The samples were handled immediately and stored frozen until analyzes. All serum samples were analyzed at the laboratory of the National Public Health Institute in Turku, Finland. Serum total cholesterol concentration was analyzed with a fully enzymatic cholesterol oxidase p-aminophenazone method (Merck, Darmstadt, Germany) with an AU510 automatic analyzer (Olympus, Hamburg, Germany) or, after January 2001, with an AU400 analyzer [32 (link)]. Serum HDL-C concentration was measured after precipitation of apoB-containing lipoprotein particles by dextran sulfate 500,000 [33 (link)]. Samples used for the insulin and glucose concentration analyzes were centrifuged immediately. Details of the insulin and glucose analyzes have been reported previously [24 (link)].

The concentrations of alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), and lactate dehydrogenase (LDH) in the plasma of mice were measured on an Olympus AU400 Analyzer.