This study focuses on the use of zebrafish retinal ganglion cells (RGCs) to investigate hydrogen peroxide levels using a genetically encoded biosensor. The biosensor, roGFP2 fused to Orp1, allows for real-time imaging of redox changes in live cells.
Take zebrafish retinal ganglion cells cultured on coated coverslips.
These cells express a genetically encoded biosensor that produces the redox-sensitive green fluorescent protein roGFP2 fused to a hydrogen peroxide-sensitive enzyme, Orp1.
Transfer the coverslip with media to a live cell imaging chamber and place the chamber on an inverted microscope.
Replace the media with serum-free media to reduce background fluorescence.
Due to low hydrogen peroxide levels, roGFP2 remains in its reduced conformation.
Excite the biosensor sequentially at 405 and 480 nm wavelengths.
Reduced roGFP2 exhibits an excitation peak at 480 nm.
Switch to a hydrogen peroxide-containing media.
Hydrogen peroxide enters the cells and reacts with Orp1, oxidizing roGFP2.
Excite the biosensor again.
Oxidized roGFP2 exhibits a fluorescence increase at 405 nm and a decrease at 480 nm.
Calculate the fluorescence intensity ratio.
An increase in the ratio indicates increased cellular hydrogen peroxide levels.
On the day of imaging, check cells under the microscope to validate growth of RGC axons. For live-cell imaging, transfer the coverslips from the culture dish to a live-cell imaging chamber. Use an inverted microscope equipped with a DIC objective, OG590 long-pass red filter, and an EM-CCD camera.
Before imaging, replace the ZFCM+ medium with ZFCM-. After positioning the cells with the 10x objective, acquire images at 60x magnification using an oil immersion objective. Use an additional 1.5x magnification.
First, acquire DIC images, then image roGFP2-Orp1 using an appropriate filter set. After taking the first set of images, exchange media with media containing different treatment solutions. Media should be changed every 30 minutes of imaging to avoid pH and osmolarity changes.
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