Neuroscience JoVE (Open Access) Citable · DOI

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Name: Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons
Uploaded: 2026-07-13
Duration: 9 min 56 s
Description: Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A ...

DOI: 10.3791/2704-v

What you'll learn

✓ Prepare and load fluorescent probes (TMRM and H2DCF-DA) into live rat cortical neurons
✓ Acquire live-cell fluorescence microscopy images to measure mitochondrial membrane potential
✓ Quantify reactive oxygen species levels in neurons using fluorescence intensity analysis
✓ Analyze and interpret changes in probe fluorescence as biomarkers of cellular stress

Protocol

Biopharma Insights We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Difficulty

advanced

Total time

~4–6 hours (including cell culture preparation, probe loading, imaging, and analysis per experimental batch)

Model organism

Rat (Rattus norvegicus) cortical neurons, primary culture

Biosafety

BSL-1

Steps

Prepare stock solutions of TMRM and H2DCF-DA

Dissolve TMRM and H2DCF-DA fluorescent probes to create concentrated stock solutions. These solutions will be diluted to working concentrations for cell loading.

▶ 02:23

Load rat cortical neurons with TMRM and H2DCF-DA

Incubate live cortical neurons with diluted fluorescent probes to allow cellular uptake. Both TMRM and H2DCF-DA will accumulate in cells and become fluorescent upon activation.

▶ 03:15

Acquire live-cell images of TMRM-loaded neurons

Perform fluorescence microscopy on TMRM-labeled neurons before and after stimulus application to measure changes in mitochondrial membrane potential (ΔΨm). Record baseline and post-stimulus fluorescence intensity.

▶ 04:42

Acquire live-cell images of H2DCF-DA-loaded neurons

Perform fluorescence microscopy on H2DCF-DA-labeled neurons to detect reactive oxygen species (ROS) accumulation. Increased fluorescence intensity indicates elevated ROS levels in response to stimulus.

▶ 06:17

Analyze fluorescence intensity changes quantitatively

Process microscopy images to extract and compare fluorescence intensity values before and after stimulus application. Calculate relative changes in TMRM and H2DCF-DA fluorescence as indicators of mitochondrial function and oxidative stress.

▶ 07:18

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