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.

NOTE: See Figure 1 for a schematic overview of key steps.
1. WST-1 Reduction Assay
<ol>
	<li>Grow and differentiate C2C12 adherent cells using standard cell culture procedures7 in a 96-well plate utilizing rows A-F.
	<ol>
		<li>Use a differentiation medium consisting of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 2% horse serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Incubate the cells at 37 &#176;C with ...

<ol>
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D., Wang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemical+and+cellular+basis+of+cell+proliferation+assays+that+use+tetrazolium+salts.">The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts.</a> Biochemica. 4, 15-20 (1996).</li><li>Tan, A. S., Berridge, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> J Immunol Methods. 238 (1-2), 59-68 (2000).</li><li>Phillips, P. 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The authors have nothing to disclose.

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...

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&#39;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.

<table>
	<tbody>
		<tr>
			<td>C2C12 myoblasts</td>
			<td>American Type Culture Collection&#160;</td>
			<td>CRL-1772</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle&#39;s medium - low glucose</td>
			<td>Sigma</td>
			<td>D6046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Plex animal serum complex</td>
			<td>Gemini Bio-Products&#160;</td>
			<td>100-602</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>516106</td>
			<td></td>
		</tr>
		<tr>
			<td>horse serum</td>
			<td>Gibco Technologies</td>
			<td>16050-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>Sigma</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm culture dishes</td>
			<td>TPP</td>
			<td>93150</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well culture plates</td>
			<td>TPP</td>
			<td>92096</td>
			<td></td>
		</tr>
		<tr>
			<td>2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Sodium Salt (WST-1)</td>
			<td>Accela ChemBio&#160; Inc</td>
			<td>SY016315</td>
			<td></td>
		</tr>
		<tr>
			<td>phenazine methosulfate&#160;</td>
			<td>Sigma</td>
			<td>P9625</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma</td>
			<td>A5960</td>
			<td></td>
		</tr>
		<tr>
			<td>ascorbate oxidase&#160;</td>
			<td>Sigma</td>
			<td>A0157</td>
			<td></td>
		</tr>
		<tr>
			<td>superoxide dismutase&#160;</td>
			<td>Sigma</td>
			<td>S5395</td>
			<td></td>
		</tr>
		<tr>
			<td>2,6-dichloroindophenol sodium salt&#160;</td>
			<td>ICN Biomedicals</td>
			<td>215011825</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES sodium salt</td>
			<td>Sigma</td>
			<td>H3784</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium chloride</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP366</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>M5921</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chloride dihydrate</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium phosphate</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP363</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Powerwave X-I spectrophotometer</td>
			<td>Biotek Insturments</td>
			<td>discontinued&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectronic Genesys 5 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>336001</td>
			<td></td>
		</tr>
		<tr>
			<td>PureGrade 96-well microplate, F-bottom, clear, untreated, non-sterile</td>
			<td>MidSci</td>
			<td>781602</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (II) chloride tetrahydrate</td>
			<td>Sigma</td>
			<td>220299</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (II) sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>215422</td>
			<td></td>
		</tr>
		<tr>
			<td>hypoxanthine</td>
			<td>Sigma</td>
			<td>H9636</td>
			<td></td>
		</tr>
		<tr>
			<td>xanthine oxidase</td>
			<td>Sigma</td>
			<td>X4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>R Studio</td>
			<td>Rstudio</td>
			<td></td>
			<td>https://www.rstudio.com/products/rstudio/</td>
		</tr>
		<tr>
			<td>KC4</td>
			<td>Biotek Insturments</td>
			<td>discontinued&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring trans-plasma membrane electron transport by c2c12 myotubes

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.

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...

Watch this Scientific Journal Video about Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes at JoVE.com

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

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.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

The overall goal of this procedure is to monitor the process of trans-plasma membrane electron transport in real time through spectrophotometric assays. This method can help answer key questions in the radox biology field regarding the mechanism of trans-plasma membrane electron transport and how a cell's redox environment is maintained. The main advantage of this technique is that the assay is a real time, multi-well, quick spectrophotometric assay.To begin, seat C2C-12 adherence cells at two times 10 to the 4th cells per square centimeter in rows A through F of a 96 well plate, then cover the cells with growth media. Incubate the cells in a 37 degree Celsius incubator with 5%carbon dioxide. After 24 hours in culture, aspirate the growth media, wash with 150 microliters of PBS, replace with differentiation medium, and incubate the cells for an additional 24 to 48 hours.Next, prepare a stock WST-1 and PMS solutions. Make a 10 millimolar stock solution of WST-1 by dissolving 033 grams of WST-1 in five milliliters of PBS. Then, vortex the solution.Then, prepare the stock solution of PMS by dissolving 0023 grams of PMS in 1.5 milliliters of the ionized water. Vortex the solution. To prepare the working solution, add glucose to PBS to make 11.4 milliliters of a five millimolar glucose solution.To this solution, add 480 microliters of the WST-1 stock solution for a final concentration of 400 micromolar WST-1 and 48 microliters of the PMS stock solution for a final concentration of 20 micromolar PMS. Vortex the solutions to mix. Then divide the solution into two six milliliter aliquots.Depending on the assay, add the relevant components described here. With the solutions now prepared, aspirate the differentiation media from the cells and wash them once with 150 microliters of PBS. Remove the PBS and add 100 microliters from aliquot one into columns one through six.And add 100 microliters from aliquot two to columns seven through 12 of the 96 well plate. Place the 96 well plate into a plate reader and measure the absorbance values every 10 minutes for one hour at 438 nanometers. After the final reading, aspirate the media and wash each well with 150 microliters of PBS.Then, aspirate the PBS. For analysis, calculate the change in absorbance for each well by subtracting the initial absorbance for the well from the absorbance at any later time point for that same well. Correct this change based on the change in absorbance that is observed in the background wells.Once background corrected, normalize the absorbance data to the 60 minute control measurements. Alternatively, normalize by the protein content in each well using a standard BCA protein assay. Finally, quantify the data to obtain the amount of WST-1 reduction by microgram of protein.To begin the DPIP reduction assay, grow and differentiate C2C-12 adherence cells, as shown for the WST-1 reduction assay. Next, prepare a stock solution containing 10 millimolar DPIP by dissolving 029 grams of DPIP in 10 milliliters of the ionized water. Then vortex the solution.Confirm the concentration by measuring the solution's absorbance at 600 nanometers using a spectrophotometer. To make the working solution, add 120 microliters of the 10 millimolar DPIP stock solution to 11.88 milliliters of a five millimolar solution of glucose and PBS for a final DPIP working concentration of 100 micromolar. Then, vortex the solution.Then divide the solution into two six milliliters aliquots. Depending on the assay, add the relevant components described here. With the solutions now prepared, aspirate the differentiation media from the cells and wash them once with 150 microliters of PBS.Remove the PBS and add 100 microliters from aliquot one into columns one through six, and 100 microliters from aliquot two to columns seven through 12 of the 96 well plate. Place the 96 well plate into a plate reader and measure the absorbance values every 10 minutes for one hour at 600 nanometers and analyze the resulting values as described in the WST-1 reduction assay analysis. To begin, prepare two solutions.First, prepare six milliliters of 400 micromolar WST-1 and 20 micromolar PMS in PBS. Then prepare six milliliters of a 100 micromolar solution of DPIP and PBS. Prepare the first 96 well plate by adding 100 microliters of a WST-1/PMS solution to rows A through D in a flat bottom plate in the absence of cells, and measure the absorbance in a spectrophotometer at 438 nanometers.Then prepare a second 96 well plate by adding 100 microliters of the DPIP solution to rows A through D in a flat bottom plate in the absence of cells, and measure the absorbance in the spectrophotometer at 600 nanometers. Next, add one microliter of 10 millimolar ascorbate to half of the wells for a final concentration of 100 micromolar. Monitor the absorbance in the plate until it stabilizes.After this, repeat with DPIP. Upon stabilization, determine whether the produced electron acceptors are substrates. For ascorbate oxidase for super oxide dismutates, by adding either ascorbate oxidase or super oxide dismutates to each well and monitoring the absorbance change for one hour.To monitor transplasma membrane electron transport, ascorbate oxidase was used to block the effects of ascorbate mediated reduction and superoxide dismutase was used to block superoxide mediated reduction to determine which portion of WST-1 reduction was due to each component. With the addition of ascorbate oxidase, WST-1 reduction was suppressed by about 30%indicating that about 30%of the transplasma membrane electron transport was due to the export of ascorbate. With the addition of superoxide dismutase, WST-1 reduction was suppressed by about 70%indicating that about 70%of the transplasma membrane electron transport was due to extracellular superoxide release.To determine if reduced DPIP is a substrate for ascorbate oxidase or super oxide dismutase, DPIP levels were reduced with ascorbic acid. After 20 minutes, ascorbate oxidase was added, and the absorbance was monitored. The recovery shows that reduced DPIP is a substrate for ascorbate oxidase.When super oxide dismutase was added, DPIP was also reoxidized, albeit at a slower rate;therefore, reduced DPIP is a substrate for these enzymes and not an appropriate extracellular electron acceptor to be utilized with these enzymes. Once mastered, this technique can be completed in two hours if it is performed properly. While attempting this procedure, it's important to remember to make all reagents fresh each day.After its development, this technique paved the way for researchers in the field of redox biology to explore transplasma membrane electron transport in a variety of cell types. After watching this video, you should have a good understanding of how to measure transplasma membrane electron transport utilizing extracellular electron acceptors, as well as how to determine if the electron acceptor might be a substrate for enzymes utilized in the assay.

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Biochemistry

6.8K Views.  Saint Louis University. The overall goal of this procedure is to monitor the process of trans-plasma membrane electron transport in real time through spectrophotometric assays. This method can help answer key questions in the radox biology field regarding the mechanism of trans-plasma membrane electron transport and how a cell's redox environment is maintained. The main advantage of this technique is that the assay is a real time, multi-well, quick spectrophotometric assay.To begin, seat C2C-12 adherence cell...