Urea reagent

The levels of urea produced by CHME5 and Imhu cells were detected by the QuantiChrom Urea Assay kit (BIOassay System, Hayward, CA, USA), used according to the manufacturer’s instructions. Briefly, an aliquot of cell culture media (50 μL) was mixed with 200 μL Urea Reagent (Bioassay system) and the absorbance measured at 430 nm in a spectrophotometric microplate reader (PerkinElmer). A standard curve was generated during each assay in the range of concentrations 0–100 μg/ml using urea as standard. In this range, the detection of urea resulted linear, and the minimum detectable concentration was 3.12 μg/mL. Protein content in each sample was determined by Bradford method (Bio-Rad Laboratories) using BSA as standard.

Cells were plated at a density of 20,000 cells/well and urea levels were measured after 48 h of treatment. Urea assay was performed also with CHME-5 treated with TII for 72 h. In all these cases, urea levels were detected by the QuantiChrom Urea Assay kit (BioAssay System, Hayward, CA, USA) according to the manufacturer’s instructions. Two hundred μL Urea Reagent (BioAssay system, Hayward, CA, USA) were added to 50 μL culture medium, and the absorbance measured at 430 nm in a spectrophotometric microplate reader (PerkinElmer Inc., MA, USA) after 50 min of incubation at room temperature under slow agitation conditions. A standard curve was generated in the range of concentrations 0–100 μg/mL using Urea as standard, and all the data were normalized proteins.

Urea levels in IMhu cells were detected by the QuantiChrom Urea Assay kit (Cat. No.: DIUR-100—BioAssay System, Hayward, CA, USA), used according to the manufacturer’s instructions. Briefly after 24 h of incubation with the tested substances, an aliquot of cell culture media (50 µL) was mixed with 200 µL Urea Reagent (BioAssay system, Hayward, CA, USA) and the absorbance measured at 430 nm in a spectrophotometric microplate reader (PerkinElmer Inc., Waltham, MA, USA). A standard curve was generated during each assay in the range of concentrations 0–100 µg/mL using urea as standard. In this range, standard detection resulted linear, and the minimum detectable concentration of urea was 3.12 µg/mL. The protein content in each sample was determined by Bradford’s method (Cat. No.: 5000006—Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA—Cat. No.: A2153—Sigma-Aldrich, St.Louis, MO, USA) as standard.