Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

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 organisms^2,3,4,5. Specifically, this process has been demonstrated in Saccharomyces cerevisiae, carrot cells, erythrocytes, lymphocytes, osteosarcoma, melanoma, macrophages, skeletal muscle, and neutrophils^2,3,<....

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 c....

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.......

Materials

List of materials used in this article

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