Feb 25, 2026
Measuring Mitochondrial Membrane Potential in iNeurons Through Live-Cell TMRM Imaging
- Benjamin O'Callaghan1,2,
- Helene Plun-Favreau1,2
- 1Department of Neurodegenerative Diseases, UCL Queen Square Institute of Neurology, London, UK;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, USA, 20815
External link: https://doi.org/10.1101/2025.04.01.646648
Protocol Citation: Benjamin O'Callaghan, Helene Plun-Favreau 2026. Measuring Mitochondrial Membrane Potential in iNeurons Through Live-Cell TMRM Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn1yjqv5d/v1
Manuscript citation:
Benjamin O’Callaghan, Daniela Melandri, Darija Soltic, Katharina Cosker, Marc P.M. Soutar, Helene Plun-Favreau (2025) iNeurons are sweet, maybe too sweet? Exploring the impact of media composition on PINK1-dependent mitophagy.bioRxiv doi: 10.1101/2025.04.01.646648
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 25, 2026
Last Modified: February 25, 2026
Protocol Integer ID: 244003
Keywords: ASAPCRN, iNeurons, TMRM, Mitochondrial Membrane Potential, N2B27, BrainPhys, mitochondrial membrane potential in ineuron, measurement of mitochondrial membrane potential, measuring mitochondrial membrane potential, sensitivity of the mitochondrial membrane potential, application of mitochondrial toxin, cell imaging of tmrm, mitochondrial toxin, mitochondrial membrane potential, cell tmrm imaging protocol, cell imaging, expressing ineuron, ineuron
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP 000478
Guarantors of Brain Non-Clinical Fellowship
Grant ID: B.O'C 2025
Abstract
Protocol outlining the measurement of mitochondrial membrane potential in iNeurons and the sensitivity of the mitochondrial membrane potential to application of mitochondrial toxins, through live-cell imaging of TMRM in mitoSRAI expressing iNeurons.
Materials
Materials
- A23_pLVX_mitoSRAI expressing iNeurons cultured on 96-well PhenoPlates (see https://dx.doi.org/10.17504/protocols.io.261ged7qjv47/v1)
(iNeuron hPSC Line established on WTC11 hPSC background with TET-ON system at the AAVS1 safe-harbour locus for overexpression of murine Ngn2 and dCas9-KRAB at the CLYBL safe harbour locus was a kind gift from the lab of Michael Ward)
- Tetramethylrhodamine methyl ester perchlorate (TMRM) (Sigma, T5428)
- Oligomycin (Sigma O4876)
- Antimycin-A (Sigma A8674)
- Rotenone (Sigma R8875)
- Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP) (Sigma, C2759)
- Opera Phenix High-Content Confocal Microscope System
Troubleshooting
Safety warnings
Oligomycin, Antimycin-A, Rotenone and CCCP are mitochondrial poisons which should be handled with appropriate PPE and disposed of through safe disposal routes.
Live-Cell Imaging of TMRM and Sensitivity to Mitochondrial Toxins
For live-cell imaging of TMRM, MitoSRAI iNeurons are cultured in 96-well Phenoplates with N2B27 vs BrainPhys media, as described in "Detection of Lysosomal Delivery of Mitochondria in iNeurons Using MitoSRAI Reporter" (https://dx.doi.org/10.17504/protocols.io.261ged7qjv47/v1).
By using iNeurons expressing mitoSRAI we are able to define the YPet+ mitochondrial region to measure TMRM intensity within, which permits mitochondria with low mitochondrial membrane potential (i.e. low TMRM staining intensity) to be detected and more accurately def.
24:00:00 prior to live-cell imaging, a half media change is performed, whilst simultaneously adjusting the culture volume to 100 µL final volume per well (e.g. for cultures in 150 µL , remove 100 µL of media and add 50 µL of fresh media).
Treatment of iNeurons with Mitochondrial Toxins
Apply desired mitochondrial toxins for testing 2h prior to live-cell imaging.
Prepare a 6x concentrated [mitochondrial toxin] diluted in complete N2B27 or BrainPhys cell culture media.
Example below for a selection of mitochondrial toxins which can be informative.
| Desired Final [Mitochondrial Toxin] | 6x [Mitochondrial Toxin] for addition | |
| 0.5µM O/A | 3µM O/A | |
| 1µM O/A | 6µM O/A | |
| 5µM O/A | 30µM O/A | |
| 1µM Rotenone | 6µM Rotenone | |
| 5µM Rotenone | 30µM Rotenone | |
| 10µM CCCP | 60µM CCCP | |
| 10µM CCCP + 1µM Oligomycin | 60µM CCCP + 6µM Oligomycin |
Mitochondrial toxin dilutions and final concentrations. (O/A = equimolar combination of Oligomycin and Antimycin-A)
Apply 20 µL of the 6x mitochondrial toxin dilution prepared in onto the 100 µL of cell culture media currently on the cells.
Place in humidified incubator, 37 °C 5%/95% CO2/AIR mixture for 01:00:00
TMRM Staining
We use TMRM in redistribution mode whereby an increase in mitochondrial TMRM intensity is linked to more polarised mitochondrial membrane potential. This requires a non-saturating [TMRM], with 25 nanomolar (nM) being a suitable concentration in our hands.
Prepare a 7x concentrated = 175 nanomolar (nM) TMRM dilution in N2B27 or BrainPhys cell culture media.
After 01:00:00 of incubation with the mitochondrial toxins within apply 20 µL of the 7x TMRM prepared in onto the 120 µL of cell culture media + mitochondrial toxin currently on the cells.
Place in humidified incubator, 37 °C 5%/95% CO2/AIR mixture for 01:00:00
Measurements of TMRM Staining Intensity
Pre-equilibriate the Opera Phenix High-Content System to 37 °C and 5%/95% CO2/AIR mixture.
We typically begin this straight after application of TMRM in
After 01:00:00 of incubation with TMRM in cells are imaged live on the pre-equilibriated Opera Phenix High Content System.
Imaging Parameters on Opera Phenix:
Use 63x objective.
In confocal mode do a z-stack.
1µm thick slices throughout entirety of the cells.
Sequentially detect the 2x fluoroscent channels using 50% laser power intensities to avoid phototoxicity/bleaching.
Adjust pixel dwell time to give good signal but avoid prolonged acquisition times.
YPet: Excitation 488nm laser, Emission 500-550nm
TMRM: Excitation 561nm laser, Emission 570-630nm
I usually do 5x fields per well and will take ~40min to image 60-wells.
Note: The Opera Phenix images in a column by column pattern. To avoid influence of differences in incubation times between conditions of interest it is recommended to split image acquisition such that one well from each conditions is measured as quickly as possible. Followed by second technical replicate wells. If all conditions of interest are positioned column by column, the entire plate can be set to image at once.
Analysis
Using the columbus software the below analysis strategy gives an accurate quantification of mitochondrial membrane potential and its sensitivity to mitochondrial toxins.
- Using max intensity project
- Detect total mitochondrial region by finding YPet positive spots.
- Measure the mitochondrial TMRM intensity by quantifying mean TMRM intensity within the YPet positive spot region. This will be output as a mean intensity across all fields selected within a well.
(Note: we prefer to measure the TMRM intensities within the entire YPet region as one, rather than on a spot by spot basis to avoid confounding influences of large mitochondrial regions being underrepresented)
4. Average the mitochondrial TMRM intensity across technical replicate wells to get the average for that biological N.