Oxiselect nitrotyrosine kit

Accumulated intracellular reactive oxygen species (ROS) were quantified in vitro using the fluorescent probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA, KeyGEN, CHN), and the fluorescent intensity was then measured with a fluorescent microscope (Carl Zeiss) and flow cytometry. Data were analyzed using ImageJ and FlowJo Software. To measure the ROS level in the retina, 3-nitrotyrosine proteins were detected in retinal lysates using the Oxiselect Nitrotyrosine Kit (Cell Biolabs, Inc., USA) according to the manufacturer’s instructions. Ten thousand cells were collected and analyzed in each flow cytometric assay. Six independent assays were performed.

Inverse fluorescence microscopy (Olympus, Japan) was used to take pictures of the stained cells after they had been incubated with 2 M dihydroethidium (DHE) (Cat# D23806, Thermo-Fisher Scientific, Pittsburgh, PA, USA) for 10 min. To measure retinal ROS levels, 3-nitrotyrosine protein levels were determined in mice retinal lysates using an Oxiselect Nitrotyrosine kit (Cat#STA-305, Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions.