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 BioRender.com.

Quantification of Mitochondrial Membrane Potential and Superoxide Levels

The authors have no competing interests to declare.

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...

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&#39;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 been genetically linked to mitophagy, a mitochondrial quality control pathway necessary for maintaining cellular homeostasis that selectively removes damaged mitochondria9,10. Studies have identified multiple independent mitophagy pathways, including FUN14 domain containing 1 (FUNDC1)-mediated mitophagy, Bcl-2 interacting protein 3 (BNIP3)-facilitated mitophagy, NIX-dependent mitophagy, and the well-characterized PTEN-induced kinase 1 (PINK1)/Parkin-regulated mitophagy10,11. PINK1 (a putative kinase) and Parkin (an E3 ubiquitin ligase) work in tandem to phospho-ubiquitinate damaged mitochondria, which drives the formation of autophagosomes that engulf the damaged organelle and fuse with lysosomes to initiate degradation12,13,14,15,16. Mutations in Parkin have been associated with PD-linked phenotypes such as neurodegeneration via the loss of dopaminergic neurons17,18.
Here, a protocol is described in which HeLa cells, routinely used immortalized cells derived from cervical cancer, are used to investigate the role of Parkin in maintaining mitochondrial network health. HeLa cells express negligible levels of endogenous Parkin and therefore require exogenous Parkin expression19. To study the role of Parkin in mitochondrial network health, HeLa cells are transfected with either wild-type Parkin (ParkinWT), a Parkin mutant (ParkinT240R), or an empty control vector. ParkinT240R is an autosomal recessive juvenile parkinsonism mutation that affects Parkin E3 ligase activity, significantly reducing the efficiency of the mitophagy pathway20. HeLa cells are subject to mild (5 &#181;M) or severe (20 &#181;M) concentrations of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. Treatment with severe concentrations of CCCP is routinely used to induce Parkin-mediated mitophagy in various cell lines, such as HeLa and COS-7 cells21,22,23.
Following treatment, the protocol employs live imaging of the mitochondrial network using two currently available mitochondrial-targeted fluorescent dyes. Tetramethylrhodamine, ethyl ester, perchlorate (TMRE) is a cationic dye that fluoresces based on mitochondrial membrane potential24, while MitoSOX is a mitochondrial superoxide indicator where the fluorescence intensity is a function of superoxide concentration25. Finally, the outlined protocol uses a fluorescence-based quantification and simple workflow to effectively quantify the mitochondrial membrane potential and superoxide levels with minimal scope for user bias. Although this protocol was designed to study mitochondrial function in HeLa cells, it can be adapted for additional cell lines and primary cell types to quantitively characterize mitochondrial network health.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals, Peptides, and Recombinant Proteins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CCCP (carbonyl cyanide m-chlorophenyl hydrazone)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C2759</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (1x) with 4.5 g/L glucose</td>
			<td>Gibco</td>
			<td>11-965-084</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO, Anhydrous</td>
			<td>ThermoFisher Scientific</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH3007103</td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE 6 (Tranfection Reagent)</td>
			<td>Promega</td>
			<td>E2691</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX 100x (L-Glutamine Solution)&#160;</td>
			<td>Gibco&#160;</td>
			<td>35-050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht 33342</td>
			<td>ThermoFisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoSOX&#160; Red&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Deep Red</td>
			<td>ThermoFisher Scientific</td>
			<td>M7514</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM (Redued Serum media)</td>
			<td>ThermoFisher scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>T669</td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental models: Organisms/Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa-M (Homo sapiens)</td>
			<td>A. Peden (Cambridge Institute for Medical Research)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant DNA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EYFP Empty Vector</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>YFP-Parkin T240R</td>
			<td>This Paper</td>
			<td>Generated by site-directed mutagenesis from YFP-Parkin</td>
			<td></td>
		</tr>
		<tr>
			<td>YFP-Parkin WT</td>
			<td>Addgene; PMID:19029340</td>
			<td>RRID:Addgene_23955</td>
			<td></td>
		</tr>
		<tr>
			<td>Software and Algorithms</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Illustrator</td>
			<td>Adobe Inc.</td>
			<td>https://www.adobe.com/products/illustrator</td>
			<td>(Schindelin, 2012)</td>
		</tr>
		<tr>
			<td>Excel (Spreadsheet Software)</td>
			<td>Microsoft Office&#160;</td>
			<td>https://www.microsoft.com/en-us/microsoft-365/excel</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td>https://imagej.net/software/fiji/</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite (LAS X)</td>
			<td>Leica</td>
			<td>https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft Office</td>
			<td>https://www.microsoft.com/excel</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism9 (Statistical Analysis Software)</td>
			<td>GraphPad Software</td>
			<td>https://www.graphpad.com</td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Dish, No. 1.5 Coverslip, 20 mm Glass Diameter, Uncoated</td>
			<td>MatTek</td>
			<td>P35G-1.5-20-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Cage Incubator (Environmental Chamber)</td>
			<td>Okolab</td>
			<td>https://www.oko-lab.com/cage-incubator</td>
			<td></td>
		</tr>
		<tr>
			<td>DMiL Inverted Microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>LIGHTNING Deconvolution Software</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>STELLARIS 8 confocal microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence-based quantification of mitochondrial membrane potential and superoxide levels using live imaging in hela cells

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.

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

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

This research focuses on mitochondria dysregulation in neurodegenerative diseases. We believe that this research can be used to understand potential causes for disease onset that could lead to therapeutics combating neurodegenerative diseases like Parkinson's disease and ALS. Techniques such as super resolution microscopy like STED and SIM or expansion microscopy have improved the ability to accurately understand protein distribution within individual organelles and mitochondria distribution throughout the cell.Results of this technique can be used as a starting point to study the effect of Parkinson's disease linked mutations on mitochondrial turnover and begin to understand the importance of regulating reactive oxygen species levels and mitochondrial membrane potential to maintain neuronal health. Our laboratory aims to mechanistically characterize individual mitochondrial quality control pathways to understand the interplay between these pathways. By having insights into pathway dynamics, one can understand how mitochondria are maintained and how mitochondrial dysregulation contributes to neurodegenerative disease onset.

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...

Watch this Scientific Journal Video about Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells at JoVE.com

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, ...

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3.1K Views.  Duke University Medical Center. This research focuses on mitochondria dysregulation in neurodegenerative diseases. We believe that this research can be used to understand potential causes for disease onset that could lead to therapeutics combating neurodegenerative diseases like Parkinson's disease and ALS. Techniques such as super resolution microscopy like STED and SIM or expansion microscopy have improved the ability to accurately understand protein distribution within individual organelles and mitochondria distribution ...

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

Biological Samples Preparation for Confocal Microscopy, Image Acquisition Setup, and Confocal Microscopy Imaging

Begin by mixing 200 microliters of reduced serum media with two micrograms of any one plasmid DNA separately in a sterile micro centrifuge tube one. Repeat this for two other plasmids in separate tubes. Then in the new tube two, mix 200 microliters of reduced serum media with six microliters of transfection reagent and mix the contents by pipetting.Incubate the tubes for five minutes at room temperature before mixing the content of tube two to tube one. In separate tubes, repeat the mixing of the other two plasmids with a transfection reagent. Incubate the mixture for 20 minutes at room temperature.Next, drop-wise, add the transfection complexes to the HeLa cell culture-seeded imaging dishes, ensuring equal distribution across the entire dish. One hour before imaging, open the carbon dioxide tank valve and turn on the environmental controller for the microscope. Using the up and down arrows on the touch pad, adjust the temperature to 37 degrees Celsius and the carbon dioxide to 5%Press Set when complete.To adjust the laser settings, turn on the white light laser by clicking the Acquire tab and selecting Open Laser Overview. In the dialogue box, toggle the white light laser to on. Enter laser power as 85%Click the excitation control button and select maximum power from the dropdown menu.Begin the tetraethyl rhodamine ethyl ester percolate or TMRE, experiment by setting the excitation laser to 514 nanometers and the emission spectra window to 524 to 545 nanometers for YFP. Next, for MitoTracker Deep Red, set the excitation laser to 641 and the emission spectra window to 650 to 750 nanometers. Similarly, for TMRE, set the excitation laser to 555 nanometers and the emission spectra window to 557 to 643 nanometers.Begin the image acquisition setting by selecting the Acquisition tab and adjusting the format to 1024 by 1024. Adjust the speed to 600 in the dropdown menu. Then click the Line Average button and from the dropdown menu, select three.Turn bidirectional scanning on and set the phase and zoom factor to 22.61 and 1.50 respectively. Once done, select the cells based on the YFP fluorescent signal by clicking on the YFP setting one and pressing Fast Live. Then adjust the gain and intensity of YFP and then select cells based on the YFP fluorescent signal.To image the DMSO control plate in the TMRE experiment, adjust the gain and intensity of the TMRE signal setting two so that the mitochondrial network intensity is just below saturation. Keep the gain and intensity for TMRE constant. Then adjust the gain and intensity of the MitoTracker setting one so that the mitochondrial network is visible, but dim.Once the gain and intensity settings are complete, click Start to acquire an image. Acquire images of 20 cells per experimental condition.

Fluorescence Intensity Quantification of Transfected HeLa Cells Using ImageJ

To measure the fluorescence intensity of the transfected HeLa cells in ImageJ software, click file and select open. In the dialogue box, select the imaging files for the experiment and click okay. Once the bio-formats import options window appears, select split channels and click okay.In tetramethylrhodamine, ethyl ester, perchlorate, or TMRE experiments, YFP is the first channel, MitoTracker is the second channel and TMRE is the third channel. Adjust the brightness by selecting image, then adjust and then the brightness. Next, load the saved region of interest or ROI by clicking analyze, then tools, then ROI manager.In the ROI manager, click more, then open from the list and select the saved ROI. Then measure the fluorescent intensity of five random regions in a single cell by selecting the saved ROI from the ROI manager and moving the ROI to a random location within a cell. To measure the fluorescence intensity, press M on the keyboard and repeat this with four additional non-overlapping regions.When a dialogue box with the area and mean gray values appears, copy and paste the values into a spreadsheet for analysis. The results for the TMRE and MitoSOX fluorescence intensities showed that their treatment with the known uncoupling agent CCCP decreased the TMRE fluorescence intensity compared to the control conditions. In addition, 20 micromolar CCCP treatment induced superoxide production and increased the MitoSOX fluorescence intensity.In mild or five micromolar CCCP stress conditions, the expression of Parkin wild-type and Parkin T240R resulted in higher TMRE intensity compared to the empty YFP control vector. The MitoSOX intensity was lower in cells expressing Parkin wild-type and Parkin T240R compared to cells expressing the YFP control vector suggesting that Parking expression helps maintain the mitochondrial network health by preserving higher mitochondrial membrane potentials and low superoxide levels.