Cm dcfda

Intracellular ROS (H₂O₂) contents were measured by incubating cells with 5 μM CM-DCFDA (Invitrogen) at 37°C for 1 h followed by detection using flow cytometry (Beckman Coulter). Intracellular superoxide level was measured by incubating cells with 5 μM MitoSOX (Invitrogen) at 37°C for 1 h before detection by flow cytometry analysis.

C4-2 NS or PXDN knockdown cells were cultured to approximately 80% confluency. Cells were trypsinized, counted and re-suspended in 20 µM of CM-DCFDA (Invitrogen, Eugene, OR, USA) and incubated at 37 °C for 1 h. The reaction was stopped on ice and the cells were centrifuged. 1 × 10⁴ cells resuspended in PBS were plated in a black walled 96-well plate and read immediately at 485 nm excitation and 535 nm emission.

Mitochondrial ROS and intracellular ROS in BUMPT cells were determined by using Mito-SOX Red (Thermo Fisher Scientific, M36008) and by CM-DCFDA (Invitrogen, C6827), respectively. ROS in kidney tissues was measured by dihydroethidium (Thermo Fisher Scientific, D11347) staining as previously described (26 (link)). Briefly, after harvesting, kidneys were snap frozen by liquid nitrogen. 20 mm sections of the kidneys were incubated with 10 mM DHE in a humidified and dark chamber at 37°C for 30 min and then counterstained with DAPI (Sigma-Aldrich, D9542). Images of the above staining were captured with fluorescence microscopy. For quantification, the fluorescence intensity in the nuclei of proximal tubular cells within 10 random optical sections was determined with ImageJ software. At least 10 randomly selected fields per mouse and four mice for each group were examined.

Prior to treatments, cells were washed and incubated with 2 μM CM-DCFDA (Life technologies, Grand Island NY) for 10 minutes followed by a wash in PBS and then recovery in DMEM for 15 min. Cells were then treated as described in figure legend. After trypsinizing, single cells were processed by flow cytometry to detect DCFDA fluorescence and mean fluorescence intensity was calculated as % increase of vehicle control.

Cells were washed with PBS and seeded in a 96-well plate at 2.10⁵ cells per well in a 200 μL reaction volume, in the presence or absence of DPI (20 μM) (Sigma-Aldrich, Saint-Quentin-Fallavier, France). DMSO was used as a vehicle control. Cytoplasmic ROS and intracellular superoxide production was detected with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-DCFDA) (Life Technologies) and dihydroethidium (DHE) (Invitrogen, Villebon sur Yvette, France), respectively, each at a concentration of 5 μM per well. ROS production was measured kinetically for CM-DCFDA (λex = 483-14 nm, λem = 530-30 nm) and DHE (λ_ex = 490-15 nm, λ_em = 600-20 nm) every minute for 1 h at 37 °C using a CLARIOstar plate reader (BMG Labtech, Champigny-sur-Marne, France). ROS production rates (RFU/min) were calculated via the CLARIOstar data analysis software, using the slope of the curve at steady state.