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