Creatinine serum colorimetric assay kit

Urinary NGAL was quantified in 50 μL of a 1:5000 dilution of urine using the Mouse Lipocalin-2/NGAL DuoSet ELISA kit from R&D Systems (Minneapolis, MN, USA) following the manufacturer’s protocol. Serum Cystatin-C was quantified in 50 μL of 1:1000 dilutions of serum, using the Mouse Cystatin C DuoSet ELISA kit from R&D Systems (Minneapolis, MN, USA) following the manufacturer’s protocol. All samples were measured in duplicate. BUN was quantified in 50 μL of a 1:10 dilution of serum using the Urea Nitrogen (BUN) Colorimetric Detection Kit from Invitrogen (Frederick, MD, USA) following the manufacturer’s protocol. Serum creatinine was quantified in 15 μL of serum using the Creatinine (serum) Colorimetric Assay Kit from Cayman Chemicals (Ann Arbor, MI, USA) following the manufacturer’s protocol. Urinary albumin was quantified in 20–40 μL of urine; Exton solution (Cole-Parmer, Vernon Hills, IL, USA) was added to urine at a 1:1 ratio and samples were incubated for 10 min in the dark. Absorbance was read at 620 nm and values were multiplied by total volume of urine (mL) to determine albumin in mg/day.

Following 8 weeks of CmNo1 treatment, whole blood was collected via retro-orbital sinus of each mouse into heparinized microhematocrit tubes (“Assistant”-Micro-Hematocrit-tubes 563, lot no. 1311444) and left undisturbed for 30 min at room temperature. The clot was removed through centrifugation at 1500 rpm for 15 min at room temperature. The serum was transferred into polypropylene tube and stored at -80°C. In order to assess the diabetic nephropathy, SCr levels were analyzed using Creatinine (serum) Colorimetric Assay Kit (700460, Cayman Chemical). The indices were examined spectrophotometrically. Further, the kidney hypertrophy index, another renal dysfunction marker was measured by kidney weight to body weight ratio [KW/BW (%)].

The kit for measuring serum troponin i type 3, Cardiac (TNNI3) was purchased from Cloud-Clone Corp. (Cat. No. SEA478Mu), and TNNI3 levels were measured using ELISA following the manufacturer’s protocol. Creatinine levels were measured using a Creatinine (serum) Colorimetric Assay Kit (Cayman Chemical) or a Creatinine (CREA) Kit (RANDOX, Cat No, CR2336). Serum BUN levels were measured using a UREA NITROGEN DIRECT kit (Stanbio laboratory) according to the manufacture’s manual.

At necropsy blood was collected from mice by cardiac puncture and placed into EDTA-tubes to prevent coagulation. Blood Samples were centrifuged and plasma was collected and frozen at −80°C until used. Cytokines and chemokines were measured using the V-PLEX Plus Pro-inflammatory Panel1 Mouse Kit from MSD (Rockville, MD). Plasma BAFF concentrations were measured using the Mouse BAFF/BLyS/TNFSF13B Quantikine ELISA Kit from R&D Systems (Minneapolis, MN) following the manufacturers protocol. Total IgG was quantified using IgG (Total) Mouse Uncoated ELISA kit (Invitrogen) following the manufacturers protocol. Anti-ds-DNA IgG present in the kidney was quantified using Mouse Anti-dsDNA IgG Antibody Assay Kit (Chondrex, inc.) following the manufacturers protocol. Plasma creatinine was measured using the Creatinine (serum) Colorimetric Assay Kit (Cayman Chemical; Ann Arbor, MI).

Biomarkers of interest in the blood were quantified by a colorimetric assay using a relevant commercially available kit, according to the manufacturer's instructions, as follows: plasma sodium (Na⁺) concentration, a Sodium assay kit (SIGMA‐ALDRICH, St. Louis, MO, USA); blood urea nitrogen (BUN), a Urea nitrogen (BUN) colorimetric detection kit (Arbor Assays, Ann Arbor, MI, USA); plasma creatinine concentration, a Creatinine (serum) colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA); level of vasopressin in plasma, an Arg⁸‐Vasopressin ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA); and level of aldosterone in plasma, an Aldosterone ELISA kit (Enzo Life Sciences).

Creatinine levels in sera were measured with the Creatinine (serum) Colorimetric Assay kit (Cayman Chemicals, Ann Arbor, MI, USA, 7000460). Briefly, 15 µL of sera sample was loaded into duplicate wells, followed by adding the Creatinine Reaction buffer and Creatinine (serum) Color Reagent. Absorbance at 490–500 nm was measured immediately after the Color Reagent buffer was added and repeated 7 min later.