Versa microplate reader

Cell proliferation was measured colorimetrically using 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution (Cell Titer 96H; Promega, Madison, WI, USA). HK-2 cells were grown in 96-well plates at a density of 10,000 cells per well. After treatment for the indicated times, MTS solution (10 μL/well) was added to the wells, the cells were incubated for 3 h at 37°C, and absorbance was measured using the VERSA microplate reader (Turner Biosystems, Sunnyvale, CA, USA).

Lactate concentration was measured using the Lactate Colorimetric Assay Kit (Abcam, Cambridge, MA, USA). Equal number of cells were suspend in Lactate Assay Buffer and all experimental procedure was performed according to the instruction of the manufacturer. Experiment was repeated three times and absorbance was measured using the VERSA microplate reader (Turner Biosystems). For p53 inhibition, 20 μM pifithrin-α (PFT-α) (Sigma-Aldrich, MO, USA) was treated for 1 h before the cisplatin treatment and quantifications were made at indicated time points. For N-acetylcysteine (NAC) (Sigma-Aldrich), which is a powerful antioxidant, 10 μM NAC was pretreated for 1 h and then cisplatin was applied. All experiments related to PFT-α and NAC followed the same treatment process.