JoVE Logo
Authors
Contact Us

Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

Abstract

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

Introduction

The ability of purified plasma membranes to reduce electron acceptors has led to the view that the plasma membrane has an inherent redox capacity1. Previously seen in fungi, plants, and animals, tPMET is a process common to multiple organisms2,3,4,5. Specifically, this process has been demonstrated in Saccharomyces cerevisiae, carrot cells, erythrocytes, lymphocytes, osteosarcoma, melanoma, macrophages, skeletal muscle, and neutrophils2,3,4,5,6,7. In a process that transports electrons across the plasma membrane to reduce extracellular oxidants, tPMET is involved in many cellular functions including: cell growth5,8, cell viability9, iron metabolism10, cell signaling11,12,13, and protection from reactive oxygen species12,14,15. Due to tPMET's involvement in many cellular functions, an imbalance of tPMET has been hypothesized to contribute to the development of some serious health conditions, including cancer16, cardiovascular disease17, and metabolic syndrome18.

There are multiple ways to monitor the transfer of electrons across the plasma membrane, but the most widely used technique is to assess the reduction of extracellular electron acceptors through colorimetric assays. Commonly used extracellular electron acceptors are tetrazolium salts, DPIP, FeCN, and ferricytochrome c19,20. The most commonly used tetrazolium salt is a second-generation salt known as WST-119. This compound is easier to utilize in colorimetric assays compared to first generation tetrazolium salts due to two sulfonate groups, which increase its water solubility21. WST-1, in conjunction with the intermediate electron acceptor 1-methoxy-phenazine methosulfate (mPMS), is reduced in two single-electron transfer events. This reduction changes the weakly-colored oxidized form of WST-1 to a more intense, yellow formazan20,22. WST-1 has a high molar extinction coefficient of 37 x 103 M-1cm-1, leading to a high assay sensitivity21,22. DPIP is also utilized as an extracellular electron acceptor to monitor tPMET. It has been shown that DPIP can be reduced extracellularly by tPMET without the aid of intermediate electron acceptors23,24. Due to the lack of intermediate electron acceptors, DPIP can directly pick-up electrons from the plasma membrane, unlike WST-124. Similar to DPIP, FeCN has been shown to be reduced extracellularly to ferrocyanide by tPMET without the aid of intermediate electron acceptors19,24. Unlike WST-1 and DPIP, FeCN has a low molar extinction coefficient leading to a lower assay sensitivity9. Another commonly used extracellular electron acceptor to monitor tPMET is ferricytochrome c. Similar to WST-1, ferricytochrome c reduction increases with the use of intermediate electron acceptor, mPMS22. Unlike WST-1 though, the ferricytochrome c method is less sensitive due to a high background and a low molar extinction coefficient22.

Here we present a method for real-time analysis of tPMET through spectrophotometric assays. The method utilized the extracellular electron acceptors WST-1 and DPIP, as they both have a high molar extinction coefficient while being less expensive compared to the other commonly used extracellular electron acceptors such as ferricytochrome c. We utilized phenazine methosulfate (PMS) instead of mPMS they have a similar chemical makeup and PMS is far less expensive. mPMS is photochemically stable which is an important characteristic for a commercial kit that needs a long shelf life. However, we make PMS fresh for each assay, so stability should not be an issue. We also present a method to evaluate possible enzymatic interactions (see Figure 1) between the extracellular electron acceptor and enzymes that may be utilized to further characterize the process of tPMET. Specifically, the enzymes AO and SOD can be used determine which portion of tPMET is due to ascorbate transport or extracellular superoxide release, two common methods for electrons to be transported across the plasma membrane.

Protocol

NOTE: See Figure 1 for a schematic overview of key steps.

1. WST-1 Reduction Assay

  1. Grow and differentiate C2C12 adherent cells using standard cell culture procedures7 in a 96-well plate utilizing rows A-F.
    1. Use a differentiation medium consisting of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2% horse serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Incubate the cells at 37 °C with 5% CO2.
    2. When monitoring ascorbate involvement in tPMET, supplement differentiation media with 100 µM ascorbic acid. Allow cells to incubate in differentiation media for ~24-48 h.
  2. Prepare stock WST-1 and PMS solutions.
    1. To make a 10 mM stock of WST-1, dissolve 0.033 g of WST-1 (Formula Weight (FW): 651.34 g/mol) in 5 mL of phosphate buffered saline (PBS). Store at 4 °C.
    2. To make a 5 mM stock of PMS, dissolve 0.0023 g of PMS (FW: 306.34 g/mol) in 1.5 mL of deionized H2O (diH2O). Store at -20 °C and protect from light.
  3. Add 0.0108 g of glucose for a final concentration of 5 mM to 11.4 mL of PBS or HEPES buffered saline (HBS; 20 mM HEPES sodium salt, 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4, 1 mM CaCl2). Add 480 µL of 10 mM WST-1 for a final concentration of 400 µM and 48 µL of 5 mM PMS for a final concentration of 20 µM.
  4. When monitoring ascorbate involvement in tPMET, divide the solution into two 6 mL aliquots. To one aliquot, add 6 µL of diH2O and to the other aliquot add 6 µL of 2 kU/mL AO for a final concentration of 2 U/mL.
  5. When monitoring superoxide involvement in tPMET, divide the solution into two 6 mL aliquots. To one aliquot, add 55 µL of 0.1 M KPO4 buffer and to the other aliquot add 55 µL of 6.5 kU/mL SOD for a final concentration of 60 U/mL.
  6. Wash the cells with PBS. Aspirate the media and add 150 µL PBS. Then aspirate the PBS.
    NOTE: The assay is done with plated, attached cells. Thus, there is no centrifugation or use of trypsin in the washing process.
  7. Add 100 µL of WST-1 solution (-) SOD or (-) AO to columns 1-6 and add 100 µL of WST-1 solution (+) SOD or (+) AO to columns 7-12 in the 96-well plate. Use rows G and H as background controls (i.e., to monitor change in absorbance in the reagent alone in wells without cells).
  8. Measure the absorbance values using the spectrophotometer every 10 min for 1 h at 438 nm.
  9. After reading, aspirate the media and wash each well with 150 µL of PBS. Then aspirate the PBS.
  10. Calculate the change in absorbance for each well by subtracting the initial absorbance for a well from the absorbance at any time point for that well. Correct this change for the change in absorbance (if any) observed in the background wells (i.e., wells with assay solution but no cells).
  11. For analysis, normalize the absorbance data to the 60 min control measurement or wash wells with PBS or HBS and then perform a bicinchoninic acid (BCA) protein assay.
  12. Add 2 mg/mL bovine serum albumin (BSA) standard (range from 0.5-2 µL for the standard curve, depending on the degree of confluence of the cells) to the empty wells (rows G and H), then add the BCA reagent to all wells.
  13. Quantify the data via nmol of WST-1 reduction per µg of protein. Use 37 mM-1 cm-1 22 as the extinction coefficient for reduced WST-1 at 438 nm.

2. DPIP Reduction Assay

  1. Grow and differentiate C2C12 adherent cells with the same procedure as step 1.1.
  2. Prepare stock 10 mM DPIP solution as follows. Dissolve 0.029 g of DPIP (FW: 290.08 g/mol) in 10 mL of diH2O. Confirm the concentration of DPIP by measuring absorbance at 600 nm with a spectrophotometer. Use 1 mM-1 cm-1 23 as the extinction coefficient for reduced DPIP at 600 nm. Store at 4 °C.
  3. Add 0.0108 g of glucose to 11.880 mL of PBS for a final concentration of 5 mM. Add 120 µL of 10 mM DPIP for a final concentration of 100 µM.
  4. When monitoring ascorbate involvement in tPMET, divide the solution into two 6 mL aliquots. To one aliquot, add 6 µL of diH2O and to the other aliquot add 6 µL of 2 kU/mL AO for a final concentration of 2 U/mL.
  5. When monitoring superoxide involvement in tPMET, divide the solution into two 6 mL aliquots. To one aliquot, add 55 µL of 0.1 M KPO4 buffer and to the other aliquot add 55 µL of 6.5 kU/mL SOD for a final concentration of 60 U/mL.
  6. Aspirate the media and wash cells in 150 µL of PBS. Aspirate the PBS and add 100 µL of DPIP solution (-) SOD or (-) AO to columns 1-6 and add 100 µL of DPIP solution (+) SOD or (+) AO to columns 7-12 in the 96-well plate. Use rows G and H will as background controls (i.e., to monitor change in absorbance in the reagent alone in wells without cells).
  7. Measure the absorbance at 600 nm using spectrophotometer every 10 min for 1 h. Quantify the change in absorbance relative to the control at 60 min similar to steps 1.9-1.13.

3. Determination of Whether Reduced Electron Acceptors Are Substrates for AO or SOD

  1. To 5.436 mL of PBS or HBS, add 240 µL of 10 mM WST-1 for a final concentration of 400 µM and 24 µL of 5 mM PMS for a final concentration of 20 µM.
    1. For DPIP, add 60 µL of 10 mM DPIP, for a final concentration of 100 µM, to 5.940 mL of PBS or HBS.
  2. Add 100 µL of solution to each well in a flat-bottom 96-well plate in the absence of cells and measure the absorbance in a spectrophotometer at 438 nm, for WST-1, or 600 nm, for DPIP.
  3. Add 1 µL of 10 mM ascorbate to half of the wells for a final concentration of 100 μM. Monitor the absorbance until it stabilizes.
  4. Upon stabilization, add 1 µL of 200 U/mL AO for a final concentration of 2 U/mL to each well or add 1 µL of 6 kU/mL SOD for a final concentration of 60 U/mL to each well and monitor the absorbance for 1 h.

Results

Statistics were performed with ANOVA with repeated measures using RStudio statistical software25. Sample sizes are indicated in the figure legends.

To monitor tPMET, C2C12 myotubes were utilized along with extracellular electron acceptors, WST-1 and DPIP. AO was used to determine which portion of WST-1 and DPIP reduction was due to ascorbate efflux and SOD was used to determine which portion of WST-1 redu...

Discussion

We have presented two methods for utilizing extracellular electron acceptors, WST-1 and DPIP, in spectrophotometric assays to monitor tPMET in C2C12 myotubes. With the growth of cell lines in standard culture procedures and a spectrophotometer plate reader, it is possible to monitor tPMET with these electron acceptors in a simple microplate assay. WST-1 reduction is reproducible from well-to-well within an assay, but there is day-to-day variability. The day-to-day coefficient of variation (CV) utilizing PBS as the buffer...

Disclosures

The authors have nothing to disclose.

Acknowledgements

We would like to thank Thomas Bell, Lyn Mattathil, Mark Mannino, and Neej Patel for their technical support. This work was supported by United States Public Health Service award R15DK102122 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) to Jonathan Fisher. The manuscript content is solely the responsibility of the authors and does not necessarily represent the official views of the NIDDK or the National Institutes of Health.

Materials

NameCompanyCatalog NumberComments
C2C12 myoblastsAmerican Type Culture Collection CRL-1772
Dulbecco's modified eagle's medium - low glucoseSigmaD6046
Fetal Plex animal serum complexGemini Bio-Products 100-602
penicillin-streptomycinSigma516106
horse serumGibco Technologies16050-130
Dulbecco's phosphate buffered salineSigmaD8537
trypsin-EDTASigmaT4049
15 cm culture dishesTPP93150
96 well culture platesTPP92096
2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Sodium Salt (WST-1)Accela ChemBio  IncSY016315
phenazine methosulfate SigmaP9625
L-ascorbic acidSigmaA5960
ascorbate oxidase SigmaA0157
superoxide dismutase SigmaS5395
2,6-dichloroindophenol sodium salt ICN Biomedicals215011825
D-(+)-glucoseSigmaG7528
HEPES sodium saltSigmaH3784
sodium chlorideSigmaS7653
potassium chlorideFisher Scientific BP366
magnesium sulfate heptahydrateSigmaM5921
calcium chloride dihydrateSigmaC7902
potassium phosphateFisher Scientific BP363
Pierce BCA Protein Assay KitThermo Scientific23225
Powerwave X-I spectrophotometerBiotek Insturmentsdiscontinued 
Spectronic Genesys 5 SpectrophotometerThermo Scientific336001
PureGrade 96-well microplate, F-bottom, clear, untreated, non-sterileMidSci781602
Iron (II) chloride tetrahydrateSigma220299
Iron (II) sulfate heptahydrateSigma215422
hypoxanthineSigmaH9636
xanthine oxidaseSigmaX4500
ExcelMicrosoft
R StudioRstudiohttps://www.rstudio.com/products/rstudio/
KC4Biotek Insturmentsdiscontinued 

References

  1. Kilberg, M. S., Christensen, H. N. Electron-transferring enzymes in the plasma membrane of the Ehrlich ascites tumor cell. Biochemistry. 18 (8), 1525-1530 (1979).
  2. Crane, F. L., Roberts, H., Linnane, A. W., Low, H. Transmembrane ferricyanide reduction by cells of the yeast Saccharomyces cerevisiae. J Bioenerg Biomembr. 14 (3), 191-205 (1982).
  3. Craig, T. A., Crane, F. L. Evidence for trans-plasma membrane electron transport system in plact cells. Proc. Indiana Acad. Sci. 90, 150-155 (1981).
  4. Mishra, R. K., Passow, H. Induction of intracellular ATP synthesis by extracellular ferricyanide in human red blood cells. J Membr Biol. 1 (1), 214-224 (1969).
  5. Crane, F. L., Sun, I. L., Clark, M. G., Grenbing, C., Low, H. Transplasma-membrane redox systems in growth and development. Biochim Biophys Acta. 811, 233-264 (1985).
  6. Berridge, M. V., Tan, A. S. Trans-plasma membrane electron transport: a cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1. Protoplasma. 205 (1-4), 74-82 (1998).
  7. Eccardt, A. M., et al. Trans-plasma membrane electron transport and ascorbate efflux by skeletal muscle. Antioxidants. 6 (4), 89 (2017).
  8. Sun, I. L., Navas, P., Crane, F. L., Morre, D. J., Low, H. NADH diferric transferrin reductase in liver plasma membrane. J Biol Chem. 262 (33), 15915-15921 (1987).
  9. Larm, J. A., Vaillant, F., Linnane, A. W., Lawen, A. Up-regulation of the plasma membrane oxidoreductase as a prerequisite for the viability of human Namalwa rho 0 cells. J Biol Chem. 269 (48), 30097-30100 (1994).
  10. Inman, R. S., Coughlan, M. M., Wessling-Resnik, M. Extracellular ferrireductase activity of K562 cells is coupled to transferrin-independent iron transport. Biochemistry. 33, 11850-11857 (1994).
  11. Castillo-Olivares, A., Esteban del Valle, A., Marquez, J., Nunez de Castro, I., Medina, M. A. Ehrlich cell plasma membrane redox system is modulated through signal transduction pathways involving cGMP and Ca2+ as second messengers. J Bioenerg Biomembr. 27 (6), 605-611 (1995).
  12. Medina, M. A., del Castillo-Olivares, A., Nunez de Castro, I. Multifunctional plasma membrane redox systems. Bioessays. 19 (11), 977-984 (1997).
  13. Medina, M. A., del Castillo-Olivares, A., Schweigerer, L. Plasma membrane redox activity correlates with N-myc expression in neuroblastoma cells. FEBS Lett. 311 (2), 99-101 (1992).
  14. Diaz-Gomez, C., Villalba, J. M., Perez-Vicente, R., Crane, F. Ascorbate Stabilization Is Stimulated in rho(0)HL-60 Cells by CoQ10 Increase at the Plasma Membrane. Biochem Biophys Res Commun. 234, 79-81 (1997).
  15. Navarro, F., et al. Protective role of ubiquinone in vitamin E and selenium-deficient plasma membranes. Biofactors. 9 (2-4), 163-170 (1999).
  16. Herst, P. M., Berridge, M. V. Cell surface oxygen consumption: a major contributor to cellular oxygen consumption in glycolytic cancer cell lines. Biochim Biophys Acta. 1767 (2), 170-177 (2007).
  17. Baoutina, A., Dean, R. T., Jessup, W. Trans-plasma membrane electron transport induces macrophage-mediated low density lipoprotein oxidation. FASEB J. 15 (9), 1580-1582 (2001).
  18. Furukawa, S., et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest. 114 (12), 1752-1761 (2004).
  19. Del Principe, D., Avigliano, L., Savini, I., Catani, M. V. Trans-plasma membrane electron transport in mammals: functional significance in health and disease. Antioxid Redox Signal. 14 (11), 2289-2318 (2011).
  20. Berridge, M. V., Tan, A. S. High-Capacity Redox Control at the Plasma Membrane of Mammalian Cells Trans-Membrane, Cell Surface, and Serum NADH-Oxidases. Antioxidants & Redox Signaling. 2 (2), 231-242 (2000).
  21. Ishiyama, M., Shiga, M., Sasamoto, K., Mizoguchi, M., He, P. G. A New Sulfonated Tetrazolium Salt That Produces a Highly Water-Soluble Formazan Dye. Chemical & Pharmaceutical Bulletin. 41 (6), 1118-1122 (1993).
  22. Berridge, M. V., Herst, P. M., Tan, A. S. Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. Biotechnology Annual Review. 11, 127-152 (2005).
  23. Gurtoo, H. L., Johns, D. G. On the interaction of the electron acceptor 2,6-dichlorophenolindophenol with bovine milk xanthine oxidase. J Biol Chem. 246 (2), 286-293 (1971).
  24. Tan, A. S., Berridge, M. V. Distinct trans-plasma membrane redox pathways reduce cell-impermeable dyes in HeLa cells. Redox Rep. 9 (6), 302-306 (2004).
  25. . . R: A language and environment for statistical computing. , (2013).
  26. Peskin, A. V., Winterbourn, C. C. A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clinica Chimica Acta. 293 (1-2), 157-166 (2000).
  27. Higaki, Y., et al. Oxidative stress stimulates skeletal muscle glucose uptake through a phosphatidylinositol 3-kinase-dependent pathway. Am J Physiol Endocrinol Metab. 294 (E889-E897), (2008).
  28. Zuagg, W. Spectroscopic characteristics and some chemical propertiesof N-methylphenazinium methyl sulfate (phenazine methosulfate) and pyocyanine at the oxidation level. J Biol Chem. 239 (11), 3964-3970 (1964).
  29. Dayan, J., Dawson, C. R. Substrate specificity of ascorbate oxidase. Biochem Biophys Res Commun. 73 (2), 451-458 (1976).
  30. Bellavite, P., della Bianca, V., Serra, M. C., Papini, E., Rossi, F. NADPH oxidase of neurtrophils forms superoxide anion but does not reduce cytochrome c and dichlorophenolindophenol. FEBS. 170 (1), 157-161 (1984).
  31. Berridge, M. V., Tan, A. S., McCoy, K. D., Wang, R. The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemica. 4, 15-20 (1996).
  32. Tan, A. S., Berridge, M. V. Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents. J Immunol Methods. 238 (1-2), 59-68 (2000).
  33. Phillips, P. A., et al. Myricetin induces pancreatic cancer cell death via the induction of apoptosis and inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Cancer Letters. 308 (2), 181-188 (2011).
  34. Altundag, E., et al. Quercetin-induced cell death in human papillary thyroid cancer (B-CPAP) cells. Journal of thyroid research. 2016 (8), (2015).
  35. Ukeda, H., Kawana, D., Maeda, S., Sawamura, M. Spectrophotometric Assay for Superoxide Dismutase Based on the Reduction of Highly Water-soluble Tetrazolium Salts by Xanthine-Xanthine Oxidase. Biosci Biotechnol Biochem. 63 (3), 485-488 (1999).
  36. Halaka, F. G., Babcock, G. T., Dye, J. L. Properties of 5-methylphenazinium methyl sulfate. Reaction of the oxidized form with NADH and of the reduced form with oxygen. J Biol Chem. 257 (3), 1458-1461 (1982).
  37. Maghzal, G. J., Krause, K. H., Stocker, R., Jaquet, V. Detection of reactive oxygen species derived from the family of NOX NADPH oxidases. Free Radic Biol Med. 53 (10), 1903-1918 (2012).

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

C2C12 MyotubesTrans plasma Membrane Electron TransportSpectrophotometric AssayWST 1PMSGlucosePBSReal time MonitoringRedox BiologyCell CultureDifferentiationAbsorbance Measurement

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Contact Us

Recommend to library

JoVE NEWSLETTERS

Research

Education

Authors

Librarians

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved