Detect x serum creatinine detection kit

Animals were culled in a rising concentration of CO₂. Once death was confirmed, cardiac puncture was performed to collect blood. This was clotted for 2 h and centrifuged at 2000 g for 20 min. Serum was collected and re‐centrifuged at 75 g for 4 min to remove all blood. Samples were stored at −20°C until analysis, repeat freeze‐thawing was avoided. Creatinine (Detect X Serum Creatinine Detection Kit, Arbor Assays) was assessed using the modified Jaffe Reaction. Urea (QuantiChrom Urea Assay Kit, BioAssay Systems) was assessed using a colorimetric reaction and Cystatin C (Quantikine ELISA, R&D Systems) was analyzed using an ELISA method.

To collect urine, mice were housed individually in metabolic cages (Tecniplast, Buguggiate, Italy) once a week for 24 hours (h). Despite being gradually acclimated to the metabolic cages, 24 h urine collection is stressful for mice, leading to weight loss of up to 1.5 g during the time spent in the cages. Because the mice on average weigh less than 20 g, it was necessary to omit urine collection at week five, since the long term anaesthesia for MSOT imaging is another stressful procedure for the mice. Total urine volume was measured and albumin levels were quantified using a Mouse Albumin ELISA Quantification Kit (Bethyl Laboratories, Montgomery, TX, USA) according to manufacturer’s instructions. Urinary creatinine (UCr) was quantified using a plate based colourimetric assay. Blood was collected via cardiac puncture after sacrifice, and separated into serum. Serum creatinine (SCr) and blood urea nitrogen (BUN) were quantified according to manufacturer’s instructions (Detect X Serum Creatinine Detection Kit, Arbor Assays, Ann Arbor, MI, USA; QuantiChrom Urea Assay Kit, BioAssay Systems, Hayward, CA, USA, respectively).

Blood samples were collected with a heparinized syringe before mice were sacrificed. Blood glucose level was determined using the glucose oxidase method. HbA1c level was determined using the DCA2000 HbA1c reagent kit (SIEMENS Healthcare Diagnostics, Inc., Tarrytown, NY, USA). Plasma creatinine level was determined using a Detect X Serum Creatinine Detection Kit (Arbor Assays, Ann Arbor, MI, USA). Cystatin C level was measured using a cystatin C Elisa kit (W Systems, Minneapolis, MN, USA). LPO level was measured by reacting with thiobarbituric acid (TBA) as described previously [33 (link)].

Blood plasma was obtained after centrifuging the blood samples at 900 g for 15 min at 4°C. The levels of plasma creatinine were measured using a DetectX Serum Creatinine Detection Kit (Arbor Assays, Ann Arbor, MI, USA). The plasma kidney injury molecule-1 (KIM1, MKM100, R&D Systems) and cystatin C (R&D Systems, Minneapolis, MN, USA) levels were determined using enzyme-linked immunosorbent assay (ELISA) kits. The thiobarbituric acid reaction was used to measure the levels of plasma lipid hydroperoxide (LPO) as described [40 (link)].

Serum creatinine and cystatin C levels were measured to assess renal function. The serum creatinine and cystatin C levels were measured using a DetectX^® Serum Creatinine Detection Kit (ARBOR ASSAYS, Ann Arbor, MI, USA) and MOUSE CYSTATIN C ELISA (BioVendor, Brno, Czech Republic), respectively. The protocol in the attached instruction manual was followed.