Nitric oxide assay kit

To evaluate the effects of cinacalcet on CsSR, CaMKKβ, phospho-Ser⁴²⁸ LKB1, and phospho-Thr¹⁷² AMPK expression, we performed immunofluorescence analysis with specific antibodies for CaSR, CaMKKβ, phospho-Ser⁴²⁸ LKB1, and phospho-Thr¹⁷² AMPK by using tyramide signal amplification fluorescence system and counterstained with DAPI. In addition, the total proteins of the HSCs were extracted with a Pro-Prep Protein Extraction Solution, following the manufacturer’s instructions. After incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Cell Signaling Technology, Danvers, MA), target proteins were visualized by an enhanced chemiluminescence substrate (ECL Plus, GE, Healthcare Bio-Science, Piscataway, NJ). To evaluate the anti-apoptotic effects of cinacalcet on HSCs in high-glucose medium, the number of TUNEL-positive cells was counted in 10 randomly chosen fields at a magnification of ×400. We also measured the concentration of NOx to quantify NO production in cell-culture media. The total NO₃ and NO₂ were quantified using the Nitric Oxide Assay Kit (Bio Vision, Mountain View, CA).

To evaluate oxidative DNA damage and lipid peroxidation, we measured the 24-h urinary 8-hydroxy-deoxyguanosine (8-OH-dG; OXIS Health Products, Inc., Portland, OR). The total urinary NO3⁻ and NO2⁻ excretion was quantified using the Nitric Oxide Assay Kit (Bio Vision, Mountain View, CA).

Nitric oxide was measured colorimetric using nitric oxide assay kit (Abcam, China, kit no ab65328). Collagen II was determined using Enzyme-linked Immunosorbent Assay (ELISA) for quantitative detection (Novus Biologicals kits). Matrix metalloproteinase 13 (MMP-13) was determined using in vitro SimpleStep ELISA® kit (Abcam, China, kit no. ab270216). NF-κB p65(Nuclear Factor Kappa B p65) was measured using Elabscience® ELISA kit. All assays were performed according to the manufacturer's instruction. All assays were performed three times.qRT-PCR was performed using Qiagen RNA extraction (Qiagen, GmbH, Hilden, Germany) and the iScript One-Step RT-PCR Kit with SYBR® Green, Bio-Rad) using reader Rotorgene RT- PCR system, according to manual instructions^{35 (link)}. The sequences of the primers used are presented in Table S1.

Intracellular NO levels in cells were measured using a Nitric Oxide Assay Kit (Abcam) according to the manufacturer's instructions. Briefly, cells were resuspended in warm culture medium and NO Red Dye stock solution was added into the culture medium. NO formation was induced by treating stained cells with LPS and incubating them at 37°C in a 5% CO₂ incubator for 6 or 24 h. Increase in fluorescence was monitored using a flow cytometer (BD FACSCanto™ System; BD Bioscience, San Jose, CA, USA) at excitation and emission wavelengths of 630 and 660 nm, respectively, and analyzed using the BD FACSDiva™ software (BD Bioscience).