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Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Published: May 12th, 2023



1Department of Cell Biology, Duke University Medical Center

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

The mitochondrial network is a series of interconnected organelles that play a crucial role in energy production1, innate immunity2,3, and cell signalling4,5. Mitochondrial dysregulation has been associated with neurodegenerative diseases such as Parkinson's disease (PD)6,7. PD is a progressive neurodegenerative disorder affecting dopaminergic neurons of the substantia nigra that impacts nearly 10 million people worldwide8. PD has bee....

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1. Preparation of biological samples

NOTE: Perform the following steps using sterile technique in a biosafety cabinet. Spray the surface of the cabinet and all materials with 70% ethanol.

  1. HeLa cell culturing and transfection
    1. Culture 30,000 HeLa cells in Dulbecco's modified eagle medium (DMEM) containing 4.5 g/L glucose supplemented with 10% fetal bovine serum and 1% L-glutamine solution (HeLa media; see Table of Materials). Plate th.......

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In this protocol, fluorescence-based quantification was used to measure the membrane potential and superoxide levels of the mitochondrial network following CCCP treatment (Figure 1). This workflow used HeLa cells, an immortalized cell line derived from cervical cancer. HeLa cells are routinely used to study mitochondrial biology and are relatively flat, making it easy to visualize the mitochondrial network using microscopy. To investigate the role of Parkin in maintaining mitochondrial netwo.......

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The workflow outlined here can be used to quantify mitochondrial membrane potential and superoxide levels robustly and reproducibly using fluorescence-based imaging30. There are important technical limitations to consider when designing these experiments. HeLa cells were transfected with an empty YFP vector, YFP-ParkinWT, or YFP-ParkinT240R. The empty YFP vector was used as a control to confirm that the experimental findings were specific to Parkin. For the transient transfec.......

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We thank the members of the Evans lab for their thoughtful feedback on this manuscript. This work is supported by Duke Whitehead Scholars, Duke Science and Technology Scholars, and Howard Hughes Medical Institute (HHMI) Hanna Gray Fellowship. Figure 1A was made using


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Name Company Catalog Number Comments
Chemicals, Peptides, and Recombinant Proteins
CCCP (carbonyl cyanide m-chlorophenyl hydrazone)  Sigma-Aldrich C2759
DMEM (1x) with 4.5 g/L glucose Gibco 11-965-084
DMSO, Anhydrous ThermoFisher Scientific D12345
Fetal Bovine Serum Hyclone SH3007103
FuGENE 6 (Tranfection Reagent) Promega E2691
GlutaMAX 100x (L-Glutamine Solution)  Gibco  35-050-061
Hoescht 33342 ThermoFisher Scientific 62249
MitoSOX  Red  ThermoFisher Scientific M36008
MitoTracker Deep Red ThermoFisher Scientific M7514
Opti-MEM (Redued Serum media) ThermoFisher scientific 31985070
Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE)  ThermoFisher Scientific T669
Experimental models: Organisms/Strains
HeLa-M (Homo sapiens) A. Peden (Cambridge Institute for Medical Research) N/A
Recombinant DNA
EYFP Empty Vector N/A N/A
YFP-Parkin T240R This Paper Generated by site-directed mutagenesis from YFP-Parkin
YFP-Parkin WT Addgene; PMID:19029340 RRID:Addgene_23955
Software and Algorithms
Adobe Illustrator Adobe Inc. (Schindelin, 2012)
Excel (Spreadsheet Software) Microsoft Office
Leica Application Suite (LAS X) Leica
Microsoft Excel Microsoft Office
Prism9 (Statistical Analysis Software) GraphPad Software
35 mm Dish, No. 1.5 Coverslip, 20 mm Glass Diameter, Uncoated MatTek P35G-1.5-20-C
Cage Incubator (Environmental Chamber) Okolab
DMiL Inverted Microscope Leica N/A
LIGHTNING Deconvolution Software Leica N/A
STELLARIS 8 confocal microscope Leica N/A

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