Colorimetric detection kit

Mouse blood samples were collected at the end of experiments. Serum urea, urea nitrogen (BUN), and creatinine were respectively assessed by Urea assay kit (MAK006, Sigma-Aldrich, St. Louis, MO, USA), Urea Nitrogen (BUN) colorimetric detection kit (EIABUN, Thermo Fisher Scientific, Waltham, MA), and Creatinine assay kit (MAK080, Sigma-Aldrich) according to the manufacturer’s instructions.

Mice were euthanized via intraperitoneal injection with sodium pentobarbital at 56 mg/kg body weight. Using heparinized syringes, blood was collected from the abdominal aorta and centrifuged at 3500 x g for 10 min at 4°C to separate the erythrocytes from plasma, and then stored at -80°C. Plasma was later analyzed for blood urea nitrogen (BUN) levels using a colorimetric detection kit (Thermo Fisher Scientific, Wilmington, DE). Bronchoalveolar lavage fluid (BALF) was collected from the lung as described previously (36 (link)). The left lung lobe was fixed in 10% neutral buffered formalin (NBF) (Fisher Scientific, Pittsburgh, PA) at a constant pressure of 30 cm H₂O for a minimum of 1 h, then immersed in a large volume of the fixative until further routine processing for light microscopic examination and morphometric analyses described below. Spleens were weighed, then half was stored in NBF along with one kidney until processed for histology. The caudal lung lobe and half a kidney were stored individually in RNAlater (Thermo Fisher Scientific, Wilmington, DE) at 4°C. The remainder of the right lung, half kidney, and half spleen were snap-frozen in liquid nitrogen and stored at -80°C.

Calcium content of the aortas and the kidneys was determined using the Calcium Detection Assay Kit (Abcam) following the manufacturer’s instructions and normalized to tissue weight. To quantify the calcium levels in MOVAS, cells were decalcified with HCl and the calcium content in supernatants was determined using the Calcium Detection Assay Kit (Abcam) according to the manufacturer’s instruction. Total protein was quantified using the PierceTM BCA Protein Assay Kit (Thermo Scientific) following the manufacturer’s instructions. The calcium content was normalized to the protein content.
Blood urea nitrogen (BUN) levels were measured using a colorimetric detection kit (Thermo Fisher Scientific) following the manufacturer’s instructions.
Calcium deposition was evaluated by staining the cells with Alizarin Red (Sigma Aldrich). Cells were washed with PBS, fixed in 4% paraformaldehyde for 15 min, stained with 2% Alizarin Red for 10 min at room temperature, and rinsed with distilled water. The stained cells were extracted with 10% cetylpyridium chloride (Sigma Aldrich) for 10 min. The OD was measured at 570 nm.