Aug 21, 2025
Characterization of mitochondrial membrane potential
- Ashley Seifert1,2,
- Hemendra Vekaria3,4,
- Ebenezer Aryee1,2
- 1University of Kentucky;
- 2Department of Biology.;
- 3Medical University of South Carolina;
- 4Department of Neurosurgery.
Protocol Citation: Ashley Seifert, Hemendra Vekaria, Ebenezer Aryee 2025. Characterization of mitochondrial membrane potential. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz9xxrgx1/v1
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: August 15, 2025
Last Modified: August 21, 2025
Protocol Integer ID: 224802
Keywords: ASAPCRN, mitochondria, membrane potential, JC-1, characterization of mitochondrial membrane potential, mitochondrial membrane potential, membrane potential of mitochondria, mitochondria, fibroblast, seifert lab
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020495
NIH
Grant ID: R01AR070313
Abstract
This protocol describes the procedure used by the Seifert Lab to characterize the membrane potential of mitochondria from fibroblasts using JC-1 dye.
Protocol materials
HBSSMerck MilliporeSigma (Sigma-Aldrich)Catalog #H4891
JC-1 Dye (Mitochondrial Membrane Potential Probe)Thermo FisherCatalog #T3168
250 mg FCCPbiorbytCatalog #orb385553
OligomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #495455
Seed cells on a 96-well black clear glass bottom plate at a density of ~30,000 cells per well
Culture for 24:00:00 to 48:00:00 until cells reach 80% confluency
Wash cells in 1X HBSSMerck MilliporeSigma (Sigma-Aldrich)Catalog #H4891
Add 100 µL of 2 micromolar (µM) JC-1 Dye (Mitochondrial Membrane Potential Probe)Thermo FisherCatalog #T3168 staining mix
Incubate for 00:30:00
Optional: to measure changes in mitochondrial membrane potential in response to inhibitors
Halfway through incubation, add 1.5 micromolar (µM) OligomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #495455 and 10 millimolar (mM) 250 mg FCCPbiorbytCatalog #orb385553
Incubate for remaining 00:15:00
Wash cells with warm 1X HBSSMerck MilliporeSigma (Sigma-Aldrich)Catalog #H4891
Scan plates with 485 nm excitation and 529 nm detection to quantify green fluorescence (monomeric)
Scan plates with 590 nm detection to quantify red fluorescence (aggregate)
Calculate mitochondrial membrane potential by the ratio of green to red fluorescence intensities, normalized to sample protein level (µg)