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. Author manuscript; available in PMC: 2023 Jan 19.
Published in final edited form as: Methods Mol Biol. 2022;2474:11–19. doi: 10.1007/978-1-0716-2213-1_2

Mitochondrial Membrane Potential Assay

Srilatha Sakamuru 1, Jinghua Zhao 1, Matias S Attene-Ramos 2, Menghang Xia 1,*
PMCID: PMC9850828  NIHMSID: NIHMS1861788  PMID: 35294751

Abstract

Mitochondrial function, a key indicator of cell health, can be assessed through monitoring changes in mitochondrial membrane potential (MMP). Cationic fluorescent dyes are commonly used tools to assess MMP. We used a water-soluble mitochondrial membrane potential indicator (m-MPI) to detect changes in MMP in various types of cells, such as HepG2, HepaRG, and AC16 cells. A homogenous cell-based MMP assay has been optimized and performed in a 1536-well plate format, which can be used to screen several compound libraries for mitochondrial toxicity by evaluating the effects of chemical compounds on MMP.

Keywords: Mitochondrial membrane potential (MMP), Mitochondrial membrane potential indicator (m-MPI), Mitochondrial toxicity, 1536-well plate, Mesoxalonitrile 4-trifluoromethoxyphenylhydrazone (FCCP)

1. Introduction

Mitochondria, commonly referred to as power houses of the cell, play a vital role in cellular physiology. The majority of the cellular energy (ATP) in eukaryotic cells is generated in the mitochondria through oxidative phosphorylation [1], during which electrons are transferred from electron donors to electron acceptors through a series of redox reactions. This mitochondrial electron transport chain creates an electrochemical gradient, which can be harnessed to drive the synthesis of ATP [2]. The electrochemical gradient also generates the mitochondrial membrane potential (MMP), which is a key parameter for evaluating mitochondrial function [3].

Mitochondrial dysfunctions have been associated with various disorders such as cancer, cardiovascular diseases, diabetes, and neurodegenerative diseases [4]. The toxicity of xenobiotic compounds can have either a direct or a secondary effect on mitochondrial function. Many of these compounds reduce MMP by perturbing a variety of macromolecules in the mitochondria, and therefore altering different mitochondrial functions. A decrease in the MMP may also be linked to apoptosis [5]. Thus, these organelles are an ideal target for in vitro toxicity studies.

Several cell membrane permeable fluorescent dyes, such as 3, 3’-dihexyloxacarbocyanine iodide [DiOC6(3)], rhodamine-123 (Rh123), tetramethyl rhodamine methyl and ethyl esters (TMRM and TMRE), and JC-1, are currently available to measure changes in MMP. Based on the assay optimization of our previous study [6], we selected the water-soluble m-MPI indicator to determine mitochondrial toxicity by screening compound libraries against HepG2 cells in a 1536-well plate format. In healthy cells, m-MPI accumulates in the mitochondria as red fluorescent aggregates (emission at 590 nm). When MMP depolarizes and cells began to deteriorate after mitochondrial toxicant treatment, m-MPI aggregates are converted to green, fluorescent monomers (emission at 535 nm) and remain in the cytoplasm (Fig. 1). So, the red/green fluorescence ratio can be used in determining mitochondrial function of cells.

Fig. 1.

Fig. 1

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MMP assay principle: In the healthy cells, m-MPI dye accumulates in mitochondria as aggregates showing red fluorescence. When mitochondrial potential collapses after FCCP treatment, the m-MPI dye remains in cytoplasm with green fluorescence. Figure reproduced from Ref. [6].

MMP assay was used to profile the Tox21 chemical collection, a large collection of environmental chemicals and drug-like molecules [7] for evaluating the compound’s ability to decrease the MMP in HepG2 cells [8]. Through a mechanism-based approach, a few chemical compounds from Tox21 collection were reported as potential mitochondrial toxicants [9]. MMP assay was tested in various other cell lines including AC16 to identify protein expression changes in human cardiomyocytes when treated with several mitochondrial toxicants [10]. MMP assay was optimized in HepaRGs, which are human hepatic cells that are more convenient to run in a high throughput format than primary human hepatocytes. MMP assay can be applied to a wide variety of cells for detecting mitochondrial dysfunction that is associated with various disorders including cancer, diabetes, cardiovascular and neurodegenerative diseases [11,12].

2. Materials

2.1. Equipment

  1. Purifier Logic+ Class II, Type A2 Biosafety Cabinet for cell operations.

  2. Steri-Cult CO2 Incubator for culturing cells at 37 °C under a humidified atmosphere and 5% CO2.

  3. Multidrop™ Combi Reagent Dispenser (Thermo Scientific, Waltham, MA) for dispensing cells into 1536-well plates by using an 8-tip dispense cassette.

  4. Pintool workstation (Wako Automation, San Diego, CA) for transferring 23 nL of compounds from a compound plate to an assay plate.

  5. BioRAPTR Flying Reagent Dispenser™ (FRD) workstation (Beckman Coulter, Inc., Brea, CA) for dispensing reagent into a 1536-well plate.

  6. Blue® Washer (Blue Cat Bio, Inc., San Francisco, CA) for removing the spent medium from the wells of a 1536 assay plate.

  7. EnVision Multilabel Plate Reader (Perkin Elmer, Shelton, CT) for reading fluorescence intensity.

  8. ViewLux uHTS Microplate Imager (Perkin Elmer) for reading luminescence intensity.

  9. ImageXpress Micro Widefield High Content Screening system (Molecular Devices, Sunnyvale, CA) for imaging purposes.

2.2. Reagents/Supplies

  1. HepG2 (human hepatocellular carcinoma), and AC16 (human cardiomyocyte) cell lines from ATCC (Manassas, VA), and Millipore Sigma (St. Louis, MO) respectively and NoSpin™ HepaRG™ (human hepatic) cells from Lonza Group Ltd (Basel, Switzerland).

  2. Cell culture medium:
    1. HepG2 cells: Eagle's Minimum Essential Medium (ATCC) supplemented with 10% fetal bovine serum (FBS), and 1% Penicillin (10,000 units/mL)/Streptomycin (10,000 μg/mL) (ThermoFisher Scientific).
    2. AC16 cells: DMEM/F12 supplemented with 12.5% EmbryoMax FBS, 2 mM EmbryoMax L-glutamine, and 1% of EmbryoMax Penicillin–Streptomycin. The culture medium and components were purchased from Millipore Sigma.
    3. NoSpin™ HepaRG™ cell plating medium: 100 mL Williams’E Medium (ThermoFisher Scientific) supplemented with 0.25% Penicillin (10,000 units/mL)-Streptomycin (10,000μg/mL) and 11.8 mL HepaRG™ Thaw and Plating supplement (Lonza Group Ltd).

  3. Trypsin-EDTA (0.05%) and StemPro Accutase (ThermoFisher Scientific) dissociation reagents for HepG2 and A16 cells, respectively.

  4. Mitochondrial Membrane Potential Indicator (m-MPI) (Codex BioSolutions, Inc., Gaithersburg, MD).

  5. Mesoxalonitrile 4-trifluoromethoxyphenylhydrazone, FCCP, (positive control compound).

  6. Tetraoctyl ammonium bromide (positive control for cytotoxicity assay).

  7. Regular and collagen-coated 1536-well black wall/clear-bottom, white wall/solid-bottom and clear polystyrene microplates for MMP assay, cytotoxicity assay and compound storage, respectively.

3. Methods

3.1. Cell Culture and Maintenance

  1. Thaw HepG2 and A16 cell lines in frozen stocks in culture medium by adding 1 mL of frozen stock to 9 mL of medium and then centrifuge for 4 min at 900 rpm. The seeding density for thawing the cell lines is 2.0 X 106 cells per T-75 cm2 flask.

  2. Culture HepG2 and A16 cells at 37°C, 5% CO2 and 95% humidity in Steri-Cult CO2 Incubator.

  3. For the expansion of HepG2 and A16, aspire the culture medium and rinse the monolayer twice with Ca2+−Mg2+-Dulbecco’s phosphate-buffered saline (DPBS). Next add 4 mL of Trypsin-EDTA (HepG2) or StemPro Accutase (AC16) solutions.

  4. Detach the cells from the surface by incubating for 3-4 min at 37°C with Trypsin-EDTA or StemPro Accutase before resuspending with culture medium.

  5. Transfer the dissociated cells to a 15 mL conical tube.

  6. Centrifuge the tubes for 4 min at 900 rpm to pellet the cells.

  7. Seed the HepG2 and A16 cells at 4.0 and 2.0 X 106 cells per T-225 cm2 and T-75 cm2 flasks, respectively.

  8. Thaw NoSpin™ HepaRG™ from the frozen stock (each vial contains 8 X 106 cells) and seed HepaRG cells directly into a collagen-coated 1536-well plate.

3.2. Quantitative High-Throughput Screening (qHTS) Protocol of MMP and Cell Viability Multiplex Assay in HepG2 and A16 cells [6, 8, 10]

  1. Plate A16 and HepG2 cells at 1,000 (A16) or 2,000 (HepG2)/well in 5 μL of the culture medium into 1536-well collagen-coated (A16) or regular (HepG2) black clear-bottom plates using FDA and Multidrop Combi, respectively. (see Note 1).

  2. Incubate the assay plates overnight at 37°C for cell adhesion.

  3. Add 23 nL of test compounds and positive control to assay plates using a Pintool station.

  4. Incubate the assay plates at 37°C for 1 h or 5 h.

  5. Add 5 μL of 2X m-MPI dye-loading solution (add 10 μL of m-MPI stock solution to 5 mL of 1X m-MPI assay buffer, mix well by vortexing) to each well using FRD (see Note 2).

  6. Incubate the assay plates at 37°C for 30 min.

  7. Measure fluorescence intensity (485 nm excitation and 535 nm emission for green, fluorescent monomers, 540 nm excitation and 590 nm emission for red fluorescent aggregates) using an Envision plate reader.

  8. Express data as the ratio of 590 nm/535 nm emissions, an indicator of MMP. The positive control compound, FCCP decreases MMP in HepG2 cells with IC50 values of 44 and 116 nM for 1 and 5 h treatment respectively (Fig. 2a) and in AC16 cells with IC50 values of 0.14 and 0.15 μM for 1 and 5 h treatment respectively (Fig. 2b).

  9. Right after the MMP assay, add 2-5 μL of CellTiter-Glo reagent (Promega) to each assay well using FRD. Use tetraoctyl ammonium bromide as a positive control for the viability assay.

  10. Incubate the assay plates at room temperature for 30 min.

  11. Measure luminescence intensity using a Viewlux plate reader.

Fig. 2.

Fig. 2

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Concentration-response curves of FCCP after 1 or 5 h treatment in HepG2 (a) and AC16 (b) cells. Each value represents the mean in duplicate experiments.

3.3. Optimization of MMP Assay in NoSpin™ HepaRG™ cells

  1. Plate NoSpin™ HepaRG™ cells at 2,500/well (each vial obtained as a frozen stock contains 8 X 106 cells that can be seeded directly into a plate) in 5 μL of the culture medium containing HepaRG™ Thaw and Plating supplement into a 1536-well, black wall/clear-bottom, collagen-coated plates (see Note 1).

  2. Incubate the assay plates overnight at 37°C for cell adhesion.

  3. The next day, spin the assay plates using Blue® Washer at light spin evacuation step to discard the spent medium, before adding 5 μL of Williams’E Medium with 0.25% Penicillin (10,000 units/mL), Streptomycin (10,000 μg/mL), 12.5% HepaRG™ Pre-Induction and Tox supplement, and 0.6% HepaRG™ Induction (serum-free) supplement using an FRD (see Note 2).

  4. Incubate the assay plates at 37°C for another 72 h by replenishing 2-3 μL culture medium to the assay wells for every 24 h.

  5. After 5 days of culturing NoSpin™ HepaRG™ cells in 1536-well assay plate, discard the spent medium from the wells using Blue® Washer at light spin evacuation step and add 5 μL of the fresh culture medium into the assay plate (media components mentioned in step 3).

  6. Treat the cells with 23 nL of positive control compound (FCCP) using a Pintool station.

  7. Incubate the assay plates at 37°C for 1 h.

  8. Add 5 μL of 2X m-MPI dye-loading solution (10 μL of m-MPI stock solution added to 5 mL of 1X m-MPI assay buffer, mixed well by vortexing) to each well using FRD (see Note 3).

  9. Incubate the assay plates at 37°C for 30 min.

  10. Measure fluorescence intensity (485 nm excitation and 535 nm emission for green, fluorescent monomers, 540 nm excitation and 590 nm emission for red fluorescent aggregates) using an Envision plate reader.

  11. Express data as the ratio of 590 nm/535 nm emissions, an indicator of MMP. The positive control compound, FCCP decreases MMP with an IC50 value of 0.7 μM for 1 h treatment (Fig. 3).

Fig. 3.

Fig. 3

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Concentration-response curve of FCCP after 1 h treatment in NoSpin™ HepaRG™ cells. Each value represents the mean in duplicate experiments.

3.4. Imaging Based MMP Assay [6, 13]

  1. Plate 2000 HepG2 cells per well in 5 μL of the culture medium into a 1536-well black wall/clear bottom plate using a Multidrop Combi.

  2. Incubate the assay plates overnight at 37°C for cell adhesion.

  3. Treat cells with the test compounds and positive control (FCCP, 6.9 and 3.5 μM).

  4. Incubate the assay plates at 37°C for 1 or 5 h.

  5. After the respective incubation times, add 5 μL of 2X m-MPI dye-loading solution with 0.3 μg/ mL of Hoechst 33342 to each well using FRD.

  6. Incubate the assay plates at 37°C for 30 min.

  7. Measure the fluorescence intensities (482 nm excitation and 536 nm emission for green, fluorescent monomers; 543 nm excitation and 593 nm emission for red fluorescent aggregates; 377 nm excitation and 447 nm emission for Hoechst 33342) using an ImageXpress Screening System.

  8. Process and analyze the imaging with the MetaXpress® and PowerCore® software using the Multi Wavelength Cell Scoring algorithm. The mean of average fluorescence intensity from each positive cell is calculated per well for both green and red fluorescent colors.

  9. Express data as ratio of 593 nm/536 nm emissions.

4. Notes

  1. The clear bottom of the assay plates should not be touched as the fluorescence intensity is read from the bottom of the plate.

  2. The spent medium in the wells of the assay plates with NoSpin™ HepaRG™ cells should be replaced with the fresh culture medium on every alternating day.

  3. For proper mixing of the m-MPI dye with the buffer, allow both solutions to thaw at room temperature before mixing.

Acknowledgement

This work was supported in part by the Intramural research program of the NCATS, NIH.

References

  • 1.Chance B, Williams GR (1956) The respiratory chain and oxidative phosphorylation. Adv Enzymol Relat Subj Biochem 17:65–134. doi: 10.1002/9780470122624.ch2 [DOI] [PubMed] [Google Scholar]
  • 2.Mitchell P (1961) Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191:144–148. doi: 10.1038/191144a0 [DOI] [PubMed] [Google Scholar]
  • 3.Chen LB (1988) Mitochondrial membrane potential in living cells. Annu Rev Cell Biol 4:155–181. doi: 10.1146/annurev.cb.04.110188.001103 [DOI] [PubMed] [Google Scholar]
  • 4.Pieczenik SR, Neustadt J (2007) Mitochondrial dysfunction and molecular pathways of disease. Exp Mol Pathol 83 (1):84–92. doi: 10.1016/j.yexmp.2006.09.008 [DOI] [PubMed] [Google Scholar]
  • 5.Lemasters JJ, Qian T, He L, Kim JS, Elmore SP, Cascio WE, Brenner DA (2002) Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis, and autophagy. Antioxid Redox Signal 4 (5):769–781. doi: 10.1089/152308602760598918 [DOI] [PubMed] [Google Scholar]
  • 6.Sakamuru S, Li X, Attene-Ramos MS, Huang R, Lu J, Shou L, Shen M, Tice RR, Austin CP, Xia M (2012) Application of a homogenous membrane potential assay to assess mitochondrial function. Physiol Genomics 44 (9):495–503. doi: 10.1152/physiolgenomics.00161.2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Attene-Ramos MS, Miller N, Huang R, Michael S, Itkin M, Kavlock RJ, Austin CP, Shinn P, Simeonov A, Tice RR, Xia M (2013) The Tox21 robotic platform for the assessment of environmental chemicals--from vision to reality. Drug Discov Today 18 (15-16):716–723. doi: 10.1016/j.drudis.2013.05.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Attene-Ramos MS, Huang R, Michael S, Witt KL, Richard A, Tice RR, Simeonov A, Austin CP, Xia M (2015) Profiling of the Tox21 chemical collection for mitochondrial function to identify compounds that acutely decrease mitochondrial membrane potential. Environ Health Perspect 123 (1):49–56. doi: 10.1289/ehp.1408642 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Xia M, Huang R, Shi Q, Boyd WA, Zhao J, Sun N, Rice JR, Dunlap PE, Hackstadt AJ, Bridge MF, Smith MV, Dai S, Zheng W, Chu PH, Gerhold D, Witt KL, DeVito M, Freedman JH, Austin CP, Houck KA, Thomas RS, Paules RS, Tice RR, Simeonov A (2018) Comprehensive Analyses and Prioritization of Tox21 10K Chemicals Affecting Mitochondrial Function by in-Depth Mechanistic Studies. Environ Health Perspect 126 (7):077010. doi: 10.1289/EHP2589 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Wei Z, Zhao J, Niebler J, Hao JJ, Merrick BA, Xia M (2020) Quantitative Proteomic Profiling of Mitochondrial Toxicants in a Human Cardiomyocyte Cell Line. Front Genet 11:719. doi: 10.3389/fgene.2020.00719 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Norat P, Soldozy S, Sokolowski JD, Gorick CM, Kumar JS, Chae Y, Yagmurlu K, Prada F, Walker M, Levitt MR, Price RJ, Tvrdik P, Kalani MYS (2020) Mitochondrial dysfunction in neurological disorders: Exploring mitochondrial transplantation. NPJ Regen Med 5 (1):22. doi: 10.1038/s41536-020-00107-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Modica-Napolitano JS, Singh KK (2004) Mitochondrial dysfunction in cancer. Mitochondrion 4 (5-6):755–762. doi: 10.1016/j.mito.2004.07.027 [DOI] [PubMed] [Google Scholar]
  • 13.Attene-Ramos MS, Huang R, Sakamuru S, Witt KL, Beeson GC, Shou L, Schnellmann RG, Beeson CC, Tice RR, Austin CP, Xia M (2013) Systematic study of mitochondrial toxicity of environmental chemicals using quantitative high throughput screening. Chem Res Toxicol 26 (9):1323–1332. doi: 10.1021/tx4001754 [DOI] [PMC free article] [PubMed] [Google Scholar]

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