Creatinine colorimetric fluorometric assay kit

The creatinine content in raw beef satay was determined using the creatinine kit purchased from Sigma-Aldrich. The extraction, purification, and analysis methods were carried out according to the manufacturer’s instructions provided in the kit. Approximately 0.1 g of raw beef satay sample was added to 400 µL of creatinine buffer assay and subjected to centrifugation at 11,000 rpm or 13,257× g for 10 min at 4 °C. Approximately 4 mL of the supernatant was removed from the homogenate, transferred into Amicon^® Ultra-4 centrifugal filters (10,000 MWL) (Merck, Darmstadt, Germany), and further centrifuged at 13,000 rpm or 18,515× g for 10 min. An aliquot (40 µL) of the supernatant was transferred into an ELISA 96-well plate and the creatinine test was completed according to the manufacturer’s protocol supplied in the creatinine colorimetric assay kit. The creatinine concentrations were measured enzymatically using the creatinine colorimetric/fluorometric assay kit (BioVision Inc., Milpitas, CA, USA). The analysis was carried out in triplicates [62 (link)].

The activity of ALT in serum was measured by using the ALT Activity Assay Kit purchased from Sigma-Aldrich, according to the supplier’s protocol. Serum creatinine level was measured by using the Creatinine Colorimetric/Fluorometric Assay Kit purchased from BioVision.

Serum creatinine of Pink1^+/+ and Pink1^−/− mice at ages 4 and 24 months was measured using a Creatinine Colorimetric/Fluorometric Assay Kit (K625‐100; BioVision), as per manufacturer's instructions. The absorbance was measured at 570 nm using a microplate reader.

Albumin and creatinine were determined from spot urine collections of the mice. Albumin was measured by Mouse Albumin ELISA (Alpco, Salem, MA, USA) and creatinine with Creatinine Colorimetric/Fluorometric Assay Kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s manual.

At the end of the 16-week treatment, mice were placed in mouse metabolic cages for 24 h urine collection. Urine samples were centrifuged and stored at −80 °C. Urinary albumin and noradrenaline were measured with enzyme-linked immunosorbent assay (ELISA) kits (Mouse Albumin ELISA, #1011, Exocell, Philadelphia, PA, USA; Norepinephrine ELISA Kit, #E4360, Biovision, Milpitas, CA, USA). Urinary kidney injury molecule-1 (KIM-1) as a marker of renal injury was quantified using a mouse kim-1 Elisa kit (CL0880, Cell applications Inc, CA, USA) according to the manufacturer’s protocol. Plasma creatinine concentrations were assayed using the Creatinine Colorimetric/Fluorometric Assay Kit (K625, Biovision, Milpitas, CA, USA). Endogenous creatinine clearance is a sensitive and accurate method for assessing glomerular filtration rate (GFR). Plasma and urine creatinine levels were determined using the above-mentioned assay kit (K625, Biovision) and calculation of creatinine clearance: GFR = U[Cr] × Volume]/P[Cr] × [Time] [25 (link)]. Since the half-life of PGI2 was short, 6-keto-PGFla, its stable and inactive metabolite, was measured. A PGE2 EIA Kit-Monoclonal (#514010, Cayman Chemical Company, MI, USA) and a 6-keto-PGFla EIA Kit (#515211, Cayman Chemical Company, MI, USA) were used according to the manufacturer’s instructions.

The 2–3 day before functional studies or tissue preparation was done, individual mice were placed in metabolic cages and urine samples were collected and quantified. We determined urine creatinine using creatinine colorimetric/fluorometric assay kit (Biovision; K625‐100) according to the manufacturer's instructions. Similarly, the IL‐6 levels in the respective/indicated samples were quantified using IL‐6 quantikine ELISA kit (R&D; M6000B) according to the manufacturer's instructions.