Labassay creatinine kit

Serum triglyceride and total cholesterol levels were measured (FUJI DRI-CHEM; Fujifilm, Tokyo, Japan). Urinary glucose levels were measured using a blood glucose meter (Abbott, Japan). Urinary creatinine levels were measured using a LabAssay Creatinine Kit (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). Urinary protein levels were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The insulin in the serum and the liver-type fatty acid-binding protein (L-FABP) in the urine and serum were measured in duplicate using a commercially available enzyme-linked immunoassay kit (#10-1247-01; Mercodia AB, Uppsala, Sweden, and RFBP10; R&D system, Minneapolis, MN, USA).

We measured canine CCL17 and CCL22 concentrations in the supernatants of serum and urine samples using the canine TARC/CCL17 and MDC/CCL22 ELISA kit (Cusabio), respectively. Urinary creatinine (Cre) concentration was measured using the LabAssay Creatinine kit (Wako), and urinary CCL17 and CCL22 concentrations were expressed as pg/mg of Cre.

In order to measure urinary oxidative stress markers, mice were placed in a urine-sampling cage for 12 h at 2 and 4 months after the experiment began, and urine was collected. Urinary isoprostane levels were determined using a urinary isoprostane F_2t ELISA kit (JaICA, Fukuroi, Japan), according to the manufacturer’s instructions. The creatinine concentration of each sample was measured using a LabAssay Creatinine kit (Wako, Osaka, Japan). Urinary isoprostane levels were normalized against creatinine concentration.

Urine samples were obtained from 1-, 5-, and 12-month-old WT nude and DMD nude rats. Urinary titin was measured using a Mouse Titin N-Fragment Assay Kit (27,602, Immuno-Biological Laboratories Co., Ltd.). Urinary creatinine (Cr) concentrations were measured using a Lab Assay Creatinine kit (636–51011, Fujifilm Wako Pure Chemical Corporation Ltd.) (Maruyama et al., 2016 (link)).

Spot urine samples were collected immediately before the administration of tacrolimus on Postoperative Day 1 and on Postoperative Days 7 and 14. All urine samples were stored at −80 °C with protease inhibitor cocktail tablets (Complete Mini; Roche Diagnostics, Mannheim, Germany). Urinary creatinine levels were measured according to the Jaffe reaction using a Lab Assay Creatinine kit (Wako Pure Chemical Industries Ltd., Osaka, Japan). Urinary biomarker levels were measured using ELISA kits purchased from R&D Systems (Minneapolis, MN) (NGAL and HE4), CMIC Co., Ltd. (Tokyo, Japan) (L-FABP), and Abcam (Cambridge, UK) (MCP-1). Biomarker levels were normalized to urinary creatinine levels to adjust for changes in urine concentration.

The hair on the back of BALB/c mice was removed using a clipper, and then 50 μL of PBS (vehicle), alum (40 mg/mL aluminum hydroxide and 40 mg/mL magnesium hydroxide), or cNPs (1000 or 3000 μg/mL) was subcutaneously injected. The injection site was photographed after 24 h. After the experiment, the mice were euthanized under isoflurane anesthesia. To evaluate organ toxicity, PBS or cNPs (1000 μg/mL) were subcutaneously injected into the back of BALB/c mice every day. On day 7, the organs (heart, lung, liver, spleen, and kidney) and blood were collected, and the blood was incubated on ice overnight. The serum was collected by centrifugation at 2000 × g for 20 min. The extracted organs were fixed in 4% PFA and sectioned at 10-μm thickness using a cryostat for staining with H&E. The tissue sections were observed using a digital microscope (BZ-9000, Keyence, Osaka, Japan). Additionally, the AST, ALT, and Cre levels in the serum were measured using a transaminase CII-test Wako kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and LabAssay Creatinine kit (Wako Pure Chemical Industries, Ltd.), respectively.