Measuring Superoxide Production in Cortical Neurons

Cortical neurons (DIV 7-12) were treated with vehicle or NMDA (150 µM) in the absence or presence of G3 or G4 phosphorous dendrimers at the indicated concentrations for different lengths of time. Afterward, the cells were incubated in K–H solution containing the ROS-sensitive fluorescent dye chloro-methyl 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA 10 µM; Molecular Probes, Barcelona, Spain) or with the mitochondrial-specific superoxide-sensitive fluorescent dye MitoSOX Red (2.5 µM; Invitrogen, Carlsbad, CA, USA) for 30 min at 37 °C. After being washed twice with K–H solution, the coverslips were placed on the stage of a Nikon Eclipse TE2000-E fluorescence microscope (Nikon, Tokyo, Japan). Excitation and emission wavelengths were set at 535 nm and 635 nm for H2DCFDA fluorescence and 510 nm and 580 nm for MitoSOX Red, respectively. Samples were recorded every 15 s using a CCD camera (Hamamatsu Photonics, Shizuoka, Japan) and analyzed using the NIS Elements AR software (Nikkon, Tokyo, Japan). Recorded fluorescence for each experimental condition was fitted to the equation y = a + bx, and the slope b was taken as an index of the rate of superoxide production as previously described [57 (link)].