JoVE Encyclopedia of Experiments
Neuroscience
0 views • 2:39 min • June 17th, 2025
Take a coverslip with genetically modified primary neurons in an imaging chamber filled with an imaging buffer.
These cells express a fluorescent protein-based redox indicator in the mitochondrial matrix.
Position the chamber under a fluorescence microscope, identify the cells under visible light, then switch to fluorescence imaging.
Apply two excitation wavelengths to stimulate the indicator and measure fluorescence emission.
The fluorescence intensity ratio at these wavelengths reflects the mitochondrial oxidation state.
Introduce NMDA to activate cellular NMDA receptors, causing intracellular calcium influx.
The increase triggers mitochondrial calcium uptake, inducing ROS production and mitochondrial
oxidation.
The ROS oxidizes the indicator, altering its fluorescence and increasing the signal ratio.
Introduce oxidizing agents to oxidize the indicator fully, maximizing the signal ratio.
Remove the buffer, then introduce reducing agents to reduce the dye completely, minimizing the signal ratio.
Use the maximum and minimum signal ratios to calibrate and quantify NMDA-induced mitochondrial oxidation.
For live imaging, set the time lapse interval to 30 seconds and duration to 25 minutes. Then,
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