2.5. Cell Viability and Proliferation

Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) colorimetric assay (M-5655, Sigma-Aldrich). Briefly, HK2 cells were plated in 96-well plates (1.5 × 10⁴ cells/well) in RPMI with 10% FBS, synchronized overnight in serum-free medium, and then incubated in triplicate in medium containing hidrosmin, vehicle, 10% FBS (positive control), and 10% DMSO (negative control) for additional 24, 48 and 72 h. After treatment, the MTT solution (0.5 mg/mL) was added for 90 min, and the absorbance of the metabolized MTT was measured at λ = 570 nm in a plate reader.

For cell proliferation assay at 24, 48, and 72 h, HK2 cells were seeded onto 96-well plates at densities of 2 × 10⁴, 1 × 10⁴, and 5 × 10³ cells/well, respectively, then synchronized overnight in serum-free medium and incubated in triplicate in RPMI with 10% FBS containing hidrosmin or vehicle, using 20% FBS and 10% DMSO as positive and negative controls, respectively. After treatment, the 5-bromo-2′-deoxyuridine (BrdU) solution was added during the last 2 h, and cells were processed according to the instructions of the BrdU Cell Proliferation ELISA Kit (ab126556, Abcam).