Cytosolic ROS generation was measured using CM-H2DCF-DA (2′-7′ dichlorofluorescin diacetate) (Molecular Probes, Eugene, OR, USA). Differentiated podocytes on 12-mm coverslips were loaded with 5 μM CM-H2DCF-DA for 20 min at 37 °C, and excess dye was then washed out using KRB solution. Fluorescence intensity was measured using an inverted microscope (IX81, Olympus, Tokyo, Japan) equipped with an array laser Nipkow spinning disk (CSU10, Yokogawa Electric Corporation, Tokyo, Japan). The wavelengths for the measurement of DCF fluorescence were 490 nm for excitation and 535 nm for emission and were analyzed using Metamorph 6.1 software (Molecular Devices).
Mitochondrial superoxide generation was detected using mitoSox (Molecular Probes), a red fluorescent dye localized to mitochondria. Once it enters the mitochondria, mitoSox is specifically oxidized by superoxide and exhibits red fluorescence. mitoSox (5 μM) was used to load the differentiated podocytes cultured on 12 mm coverslips for 14 days at 37 °C. Cells were washed twice with KRB solution and the red fluorescent intensity from podocytes was measured by using the Nipkow spinning disk confocal microscopic system with wavelengths of 514 and 560 nm for excitation and emission, respectively.
Mitochondrial superoxide generation was detected using mitoSox (Molecular Probes), a red fluorescent dye localized to mitochondria. Once it enters the mitochondria, mitoSox is specifically oxidized by superoxide and exhibits red fluorescence. mitoSox (5 μM) was used to load the differentiated podocytes cultured on 12 mm coverslips for 14 days at 37 °C. Cells were washed twice with KRB solution and the red fluorescent intensity from podocytes was measured by using the Nipkow spinning disk confocal microscopic system with wavelengths of 514 and 560 nm for excitation and emission, respectively.