Hoechst 33342 staining

Production of hydroxyl radical ( Á OH) and hypochlorous acid (HClO) were measured using the OxiORANGE reagent (Goryo Chemical, Sapporo, Japan). H 2 O 2 and NO were detected by HYDROP and diaminofluorescein-FM diacetate (DAF-FM DA) (Goryo Chemical), respectively. Cells plated in a 96-well Black IsoPlate (PerkinElmer, Waltham, MA, USA) were incubated with 0.5 µM OxiORANGE, 1 µM HYDROP, or 1 µM DAF-FM DA diluted in EMEM containing FBS for 30 min at 37 °C, and then washed with 100 µL Hank's balanced salt solution (HBSS) once. Fluorescence (Ex/Em = 535/595 nm for OxiORANGE, 485/ 535 nm for HYDROP and DAF-FM DA) was measured by using FilterMax F5 (Molecular Devices, San Jose, CA, USA). Normalized values were obtained by dividing the fluorescent intensities of HYDROP by protein concentration, those of DAF-FM DA by nuclear signal, and those of Oxi-ORANGE by nuclei number determined by Hoechst 33342 staining (Dojindo). Lipid peroxide was measured using the Liperfluo reagent (Dojindo). Cells were stained with 1 µM Liperfluo reagent diluted in EMEM without FBS for 30 min at 37 °C, and then observed under an all-in-one fluorescence microscope BZ-9000 (Keyence, Osaka, Japan).

Cells were seeded in the PureCoat Amine 96-well plate (Corning Inc., Corning, NY, USA). Cell viability was determined using the viability/cytotoxicity assay kit for animal live and dead cells (Biotium, Fremont, CA, USA), according to the manufacturer's instructions. The nuclei were visualized by Hoechst 33342 staining (Dojindo, Kumamoto, Japan; 5 mg/ml). Stained cells were imaged using the Opera with a 203 water-immersion objective in confocal mode and collected with a set of 25 fields from each well. Hoechst-positive, calcein-positive, and ethidium homodimer III (EthDIII)-negative cells were counted as viable cells by using Harmony 4.6 software.