Aug 21, 2025

Public workspaceCharacterization of mitochondrial membrane potential

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vz9xxrgx1/v1
  • Ashley Seifert1,2,
  • Hemendra Vekaria3,4,
  • Ebenezer Aryee1,2
  • 1University of Kentucky;
  • 2Department of Biology.;
  • 3Medical University of South Carolina;
  • 4Department of Neurosurgery.
Ashley Seifert
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vz9xxrgx1/v1
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
ReagentHBSSMerck MilliporeSigma (Sigma-Aldrich)Catalog #H4891
ReagentJC-1 Dye (Mitochondrial Membrane Potential Probe)Thermo FisherCatalog #T3168
Reagent250 mg FCCPbiorbytCatalog #orb385553
ReagentOligomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #495455
Troubleshooting
Seed cells on a 96-well black clear glass bottom plate at a density of ~30,000 cells per well
Culture for Duration24:00:00 to Duration48:00:00 until cells reach 80% confluency

Wash cells in 1X ReagentHBSSMerck MilliporeSigma (Sigma-Aldrich)Catalog #H4891

Add Amount100 µL of Concentration2 micromolar (µM) ReagentJC-1 Dye (Mitochondrial Membrane Potential Probe)Thermo FisherCatalog #T3168 staining mix

Incubate for Duration00:30:00

Optional: to measure changes in mitochondrial membrane potential in response to inhibitors
Optional
Halfway through incubation, add Concentration1.5 micromolar (µM) ReagentOligomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #495455 and Concentration10 millimolar (mM) Reagent250 mg FCCPbiorbytCatalog #orb385553

Optional
Incubate for remaining Duration00:15:00

Optional
Wash cells with warm 1X ReagentHBSSMerck 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)