Name: measuring trans-plasma membrane electron transport by c2c12 myotubes
Uploaded: 2018-05-04T15:00:00
Description: Watch this Scientific Journal Video about Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes at JoVE.com

Vi vil gjerne takke Thomas Bell, Lyn Mattathil, Mark Mannino og Neej Patel deres kundestøtte. Dette arbeidet ble støttet av USA Public Health Service award R15DK102122 fra National Institute Diabetes og fordøyelsesenzymer og nyre sykdommer (NIDDK) til Jonathan Fisher. Manuskriptet innholdet er ansvar forfattere og representerer ikke nødvendigvis den offisielle synet av NIDDK eller National Institutes of Health.

Merk: Se figur 1 for en skjematisk oversikt over hovedtrinnene.
1. WST-1 reduksjon analysen
<ol><li>Vokse og skille C2C12 tilhenger celler ved hjelp av standard celle kultur prosedyrer7 i en 96-brønns plate utnytte rader A-F.<ol>
<li>Bruk en differensiering medium bestående av Dulbeccos endret Eagle's Medium (DMEM) med 2% hest serum, 100 U/mL penicillin og 0,1 mg/mL streptomycin. Inkuber cellene på 37 ° C med 5% CO2.</li>
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<ol>
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Vi har presentert to metoder for å utnytte ekstracellulære elektron acceptors, WST-1 og DPIP, i Spektrofotometri analyser å overvåke tPMET i C2C12 myotubes. Med veksten av linjer i standard kultur prosedyrer og en spektrofotometer plate leser er det mulig å overvåke tPMET med disse elektron acceptors i en enkel microplate analysen. WST-1 reduksjon er reproduserbare fra godt-å-brønnen i analysen, men det er daglige variasjon. Den daglige variasjonskoeffisienten (CV) utnytte PBS som bufferen er 0,18 og CV utnytte H...

Av renset plasma membraner redusere muligheten elektron acceptors har ført til visningen at plasma membranen har en iboende redoks kapasitet1. Tidligere sett i sopp, planter og dyr, er tPMET en prosess som er felles for flere organismer2,3,4,5. Spesielt har denne prosessen blitt demonstrert i Saccharomyces cerevisiae, gulrot celler, erytrocytter, lymfocytter, osteosarcoma, melanom, makrofager, skjelettlidelser muskel og nøytrofile2,3, 4 , 5 , 6 , 7. i en prosess som transporterer elektroner i plasma membranen å redusere ekstracellulære oksidanter, tPMET er involvert i mange cellulære funksjoner inkludert: celle vekst5,8, celle levedyktighet9, jern metabolisme10, celle signalisering11,12,13, og beskyttelse fra reaktive oksygen arter12,14,15. På grunn av Tpmet's engasjement i mange cellulære funksjoner, en ubalanse i tPMET har blitt antatt bidra til utviklingen av noen alvorlige helsemessige forhold, inkludert kreft16, hjerte-og karsykdommer17, og metabolske syndrom18.
Det finnes flere måter å overvåke overføring av elektroner i plasma membranen, men den mest brukte metoden er å vurdere reduksjon av ekstracellulære elektron acceptors gjennom kolorimetrisk analyser. Brukte ekstracellulære elektron acceptors er tetrazolium salter, DPIP, FeCN og ferricytochrome c19,20. Den mest brukte tetrazolium saltet er en andregenerasjons salt kjent som WST-1-19. Denne forbindelsen er enklere å bruke i kolorimetrisk analyser sammenlignet med første generasjon tetrazolium salter på grunn av to sulfonate grupper, som øke sine vannløselighet21. WST-1, er i forbindelse med middels elektron acceptor 1-metoksy-phenazine methosulfate (mPMS), redusert i to én-elektron overføre hendelser. Denne reduksjonen endres svakt-farget oksidert form av WST-1 til en mer intens, gul formazan20,22. WST-1 har en høy molar utryddelse koeffisient av 37 x 103 M-1cm-1, fører til en høy analysen følsomhet21,22. DPIP benyttes også som en ekstracellulære elektron godkjenner å overvåke tPMET. Det har vist at DPIP reduseres extracellularly ved tPMET uten hjelp av mellomliggende elektron acceptors23,24. På grunn av manglende mellomliggende elektron acceptors, DPIP kan direkte pick-up elektroner fra plasma membranen, i motsetning til WST-1-24. Lik DPIP, FeCN har vist å være redusert extracellularly til ferrocyanide med tPMET uten hjelp av mellomliggende elektron acceptors19,24. I motsetning WST-1 og DPIP har FeCN en lav molar utryddelse koeffisient fører til en lavere analysen følsomhet9. En annen vanlig ekstracellulære elektron acceptor overvåke tPMET er ferricytochrome c. ligner WST-1, ferricytochrome c reduksjon øker med bruk av mellomliggende elektron acceptor, mPMS22. I motsetning WST-1 men er metoden ferricytochrome c mindre sensitive høy bakgrunn og en lav molar utryddelse koeffisienten22.
Her presenterer vi en metode for sanntids analyse av tPMET gjennom Spektrofotometri analyser. Metoden benyttes ekstracellulære elektron acceptors WST-1 og DPIP, som begge har en høy molar utryddelse koeffisient mens blir billigere sammenlignet med andre ofte brukte ekstracellulære elektron acceptors ferricytochrome c. Vi utnyttet phenazine methosulfate (PMS) i stedet for mPMS de har en lignende kjemisk makeup og PMS er langt billigere. mPMS er photochemically stabil som er et viktig karakteristisk for en kommersiell kit som trenger lang holdbarhet. Vi gjør imidlertid PMS fersk hver analysen, så stabilitet ikke bør være et problem. Vi presenterer også en metode for å vurdere mulig enzymatisk samspillet (se figur 1) mellom ekstracellulære elektron acceptor og enzymer som kan benyttes for å karakterisere videre prosessen med tPMET. Spesielt avgjøre enzymer AO og TORV hvilken del av tPMET er ascorbate transport eller ekstracellulær superoxide utgivelse, to vanlige metoder for elektroner transporteres over plasma membranen.

<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 membran elektronet transport (tPMET) spiller en rolle i beskyttelse av celler intracellulær reductive stress samt beskyttelse mot skade ved ekstracellulære oksidanter. Denne prosessen å transportere elektroner fra intracellulær reductants til ekstracellulære oksidanter er ikke godt definert. Her presenterer vi Spektrofotometri analyser av C2C12 myotubes å overvåke tPMET utnytte de ekstracellulære elektron acceptors: vannløselige tetrazolium salt-1 (WST-1) og 2,6-dichlorophenolindophenol (DPIP eller DCIP). Gjennom reduksjon av disse elektron acceptors er vi i stand til å overvåke denne prosessen i en sanntids analyse. Med tillegg av enzymer som ascorbate oxidase (AO) og superoxide dismutase (TORV) på råolje-terminaler, kan vi finne ut hvilken del av tPMET skyldes ascorbate eksport eller superoxide produksjon, henholdsvis. Mens WST-1 ble vist å produsere stabile resultater med lav bakgrunn, kunne DPIP være nytt oksidert etter AO og TORV, som ble demonstrert med Spektrofotometri analyse. Denne metoden viser en real-time, flere godt, rask Spektrofotometri analysen med fordeler over andre metoder brukes til å overvåke tPMET, som ferricyanide (Backward) og ferricytochrome c reduksjon.

Statistikk som ble utført med VARIANSANALYSE med gjentatte measures bruker RStudio statistisk programvare25. Utvalgene angis i figur legender.
For å overvåke tPMET, ble C2C12 myotubes brukt sammen med ekstracellulære elektron acceptors, WST-1 og DPIP. AO ble brukt til å avgjøre hvilken del av WST-1 og DPIP reduksjon var ascorbate middelklasseinnbyggere og TORV ble brukt til å avgjøre hvilken del a...

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

Måle Trans-Plasma membran elektronet Transport av C2C12 Myotubes

Målet med denne protokollen er spektrofotometrisk overvåke trans-plasma membran elektronet transport utnytte ekstracellulære elektron acceptors og analysere enzymatisk interaksjoner som kan oppstå med disse ekstracellulære elektron acceptors.

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

Research

JoVE Journal

Biochemistry

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Metode for å visualisere og analysere Membran samspill proteiner av transmisjonselektronmikroskopi

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Measuring Protein Binding to F-actin by Co-sedimentation

Måling av proteinbinding til F-aktin ved co-sedimentering

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

Analysere superkomplekser av den mitokondriale elektrontransportkjede med innfødte elektroforese, in-gel-analyser og elektrolysering

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Elektrokjemiske gjenkjenning av Deuterium Kinetic isotop effekt på ekstracellulære elektronet Transport Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Analysis of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Analyse av β-Amyloid-indusert unormalt Fibrin Clot struktur av spektroskopi og skanning elektronmikroskop

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

En fluorescens svingninger spektroskopi analysen av Protein-Protein interaksjoner i celle-celle kontakter

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Analyse av proteinfolding, Transport og nedbrytning i levende celler av radioaktivt puls Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Mitokondrier og endoplasmatiske retikulum Imaging av correlative Light og Volume Electron mikroskopi

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

Interaksjoner med og membran Permeabilization av Brain mitokondrier av amyloid fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Karakterisering av membran transportører av Heterologous uttrykk i E. coli og produksjon av membran blemmer

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...