Dri chem 4000i analyzer

For clinical biochemistry tests, whole blood samples were collected from the retrobulbar venous plexus of 26 Ndst4^−/− and 27 WT mice at 9–10 weeks of age. Sera were prepared immediately after blood coagulation and analyzed by a FUJI DRI-CHEM 4000i Analyzer (Fujifilm, Tokyo, Japan) with appropriate reagent kits. The serum biochemical parameters included four metabolites, four enzymes, lipid profile, renal function profile, and two electrolytes (Supplementary Table S4).

Blood samples were taken from the facial vein and allowed to clot for 30 min at room temperature. Serum was collected after centrifugation at 10,000 rpm for 10 min at 4 °C and stored at −80 °C until use. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TCHO), and triglycerides (TGs) were measured using a Fuji Dri-Chem 4,000i analyzer (Fujifilm, Tokyo, Japan). IL-6 and IL-10 serum levels were measured using an enzyme linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA).

After collecting blood from each mouse, the blood was allowed to stand at room temperature for 30 min, and then centrifuged at 6,000 rpm at 4 °C to collect upper serum. Serum creatinine and BUN levels were analyzed by FUJI DRI-CHEM 4000i analyzer (FUJIFILM Corp., Tokyo, Japan). On the other hand, urine creatinine level was detected by Creatinine Colorimetric Detection kit (Enzo Biochem, NY) according to the manufacturer protocol. Briefly, the mouse urine was diluted with deionized water to a final 50 µl reaction volume in a 96-well plate. For each well, 100 µl of Creatinine Detection Reagent was added and incubated for 30 min. Plate-based colorimetric measurement (490 nm) was performed by Epoch microplate spectrophotometer (Biotek, VT).

Creatinine was assessed using a DriChem 4000i Analyzer (Fuji Film, Tokio, Japan) and the corresponding DriChem slides (Fuji) according the manufacturer’s instruction.
Alpha-microglobulin determination took place by an ELISA-kit according the manufacturer’s protocol (Hölzel Diagnostika, Cologne, Germany).

Six- to eight-week-old female and male C57BL/6 mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). The GNMT knockout mice in C57BL/6 background^{74 (link)}, GNMT transgenic mice in FVB/B6 background^{38 (link)} and their littermate wild-type control were generated and maintained in specific pathogen-free conditions in accordance with the regulations at the Animal Center, Kaohsiung Medical University (KMU). Mice were treated with 2 or 5 mg/kg/day AAI (Sigma-Aldrich, St. Louis, MO, USA) or corn oil (vehicle control) by intraperitoneal (i.p.) injection, 5 days per week for 3 weeks and then were humanely euthanized by CO₂. Mouse serum were collected from tail vein before and after AAI treatment. Serum levels of ALT, creatinine and BUN were detected by FUJI DRI-CHEM 4000i analyzer (FUJIFILM Corp. Tokyo, Japan). Mouse liver and kidneys were also collected to future experiments. All experiments were performed in accordance with relevant guidelines and regulations. All animal work was approved by the Institutional Animal Care and Use Committee of KMU (IACUC approval no.: 104075, 104093).

The OA-SNO and OA-control rats fasted for 12 h before the blood sample withdrawal; blood samples were taken from the rat tail vein every 4 weeks post-ACLT + MMx surgery. The blood samples were centrifuged (8000 × g for 5 min) to separate sera and stored in a −80 °C freezer prior to analysis. The serological levels of uric acid, glucose, total cholesterol (T-CHO), high-density lipoprotein (HDL), and triglyceride (TG) were measured using the FUJI DRI-CHEM 4000i analyzer (FUJIFILM Corporation, Tokyo, Japan) at the Taiwan Mouse Clinic (Academia Sinica, Taipei, Taiwan).