Detecting Hydrogen Peroxide Levels in Cultured Neurons Through Live-Cell Fluorescence Imaging

1. In vitro reactive oxygen species (ROS) imaging of cultured retinal ganglion cell (RGC) neurons

1. On the day of imaging (typically 6-24 h after cell plating), check retinal ganglion cells under a microscope to validate the growth of RGC axons.
2. For live-cell imaging, transfer coverslips from the culture dish to a live cell imaging chamber.
3. Set up microscope for imaging. Use an inverted microscope equipped with a differential interference contrast (DIC) objective, OG590 long-pass red filter, and an EM-CCD camera.
4. Before imaging, replace the zebrafish cell culture medium with serum and antibiotics (ZFCM)(+)) with zebrafish cell culture medium without serum and antibiotics (ZFCM(-)).
5. Once cells are positioned with 10x objective, acquire images at 60x magnification using a high NA oil immersion objective. Use an additional 1.5x magnification.
6. First, acquire DIC images. Then, image roGFP2-Orp1 using an appropriate filter set. Excite roGFP2-Orp1 with 405/20 and 480/30 nm excitation filters sequentially and acquire images with 535/30 nm emission filter after emission light has passed the dichroic mirror 505DCXR.
7. After taking the first set of images, exchange media with media containing different treatment solutions. The media should be changed every 30 minutes of imaging to avoid pH and osmolarity changes.