Name: a flow cytometry-based assay for measuring mitochondrial membrane potential in cardiac myocytes after hypoxia/reoxygenation
Uploaded: 2018-07-13T13:00:00
Description: Watch this Scientific Journal Video about A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation at JoVE.com

Denne studien ble støttet av tilskudd fra The National nøkkelen forskning og utvikling Program i Kina (nr. 2017YFC1700503), National grunnleggende forskningsprogram (973 Program) i Kina (No.2012CB518602), den nasjonale Natural Science Foundation i Kina (nr. 81370223 og nr. 81573957), og Postgraduates' nyskapende Research Foundation Peking Union Medical College (2016-1002-01-02).

1. forberedelse av reagenser og løsninger
<ol><li>Forberede menneskelige hjerte myocytter (HCMs) komplett medium ved å legge 25 mL av Myocyte vekst Medium supplement til 500 mL Myocyte vekst Medium henhold til produsentens instruksjoner. Butikken på 4 ° C og varmt 37 ° c før bruk.</li>
	<li>Forberede Tongxinluo (TXL) løsning av oppløsende TXL Ultrafin pulver i serum glukose-fri Dulbeccos endret Eagle's medium (DMEM) og justere konsentrasjonen av TXL til 400 µg/mL ved å legge DMEM (for mer informasjon, se<sup ...

<ol>
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Her presenterer vi en protokoll som bruker JC-1 fargestoff for å vurdere MMP celleområde etter å bli utsatt for H/R. oppdaget av JC-1 analysen, MMP celler er uavhengig av faktorer som mitokondrie størrelse, form og tetthet som kan påvirke single-komponent fluorescens signaler14. Følgelig er resultatene av JC-1 analysen relativt pålitelig. Dessuten, det er praktisk og tidsbesparende å utføre JC-1 analysen. Denne analysen har en lav innloggings materialer reagenser, og vanligvis tar det min...

Koronar hjertesykdom er den ledende dødsårsaken som er over hele verden. Behandling av valg er for å redusere iskemiske skader og størrelsesbegrensing betennelsessykdommer hos pasienter med ST-segmentet forhøyet hjerteinfarkt rettidig og effektiv hjerteinfarkt reperfusion via primære PCI-behandling (PCI)1, 2. Imidlertid forårsaker reperfusion ytterligere skade, som kan utgjøre opptil 30 prosent av den endelige betennelsessykdommer størrelse3. Det er allment anerkjent at mitokondrie permeabiliteten overgangen pore (mPTP) ikke er bare sentral i mitokondrie skade og celle død under iskemi/reperfusion (jeg / R), men er også en konvergerende målet for Kardioprotektive signalering4 , 5. som mPTP åpning fører til depolarization av indre mitokondrie membran potensial (MMP)4, vi oppdaget mPTP åpning bruker 5, 5 ', 6, 6 '-Tetrachloro-1, 1, 3, 3-tetraethyl-imidacarbocyanineiodide (JC-1) analysen.
JC-1 analysen er en cytofluorimetric metode som er både kvalitativ og kvantitativ, og det videre er validert ved å analysere MMP på nivå med en enkelt mitokondrier6. JC-1 eksisterer som en samlet form, gir en rød til oransje fargede utslipp (590 ± 17,5 nm) i matrisen av mitokondrier med normal MMP; med tapet av MMP konverteres JC-1 til skjemaet monomerisk som gir grønne fluorescens med et utslipp av 530 ± 15 nm. Derfor en nedgang i rød/grønn fluorescens intensitet forholdet kan indikere reduksjon av MMP i forhold som iskemi/reperfusion (jeg / R).
I tillegg til JC-1, MMP også har vært studert med membran-permeable lipofile kasjoner som Rhodamine 123 og 3, 3-dihexyloxadicarbocyanine iodide [DiOC6(3)]. Men er i forhold til disse to sonder, JC-1 mer pålitelig for å analysere MMP. Rhodamine 123 har relativt dårlig følsomhet (spesielt i slukke modus7,8) og dårlig spesifisitet. Skifte i rhodamine 123 er noen ganger så liten at det er vanskelig for forskere eller utstyr å observere/oppdage. Dessuten, i en enkelt celle, det er ulike mitokondrie bindende områder for rhodamine 123 og så at det kan ha forskjellige fluorescens utslipp9. DiOC6(3) anbefales ikke for å oppdage MMP slik det reagerer følsomt depolarization plasma membranen10.
Derfor bruker her vi JC-1 analysen for å vurdere MMP HCMs etter å bli utsatt for hypoksi/reoxygenation med eller uten en beskyttende agent.

<table border="0" cellpadding="0" cellspacing="0" width="970">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">Mitochondrial membrane potential assay kit with JC-1</td>
			<td style="width:307px;">Beyodtime, China</td>
			<td style="width:80px;">C2006</td>
			<td style="width:291px;">In the kit there are JC-1 stock solution (200&#215;), stock staining buffer (5&#215;) and CCCP(10mM)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tongxinluo ultrafine powder</td>
			<td>Shijiazhuang Yiling Pharmaceutical Co., China</td>
			<td>071201</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">Annexin V-FITC/PI Kit</td>
			<td>Becton-Dickinson, USA</td>
			<td>556547</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM</td>
			<td>Life Technologies, Grand Island Biological Company, USA</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Human cardiac myocyte</td>
			<td>Promocell, Germany</td>
			<td>C-12810</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">Myocyte Growth Medium 
			(SupplementMix)</td>
			<td>Promocell, Germany</td>
			<td>C-39275</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Myocyte Growth Medium (Ready-to-use)</td>
			<td>Promocell, Germany</td>
			<td>C-22070</td>
			<td>used with Myocyte Growth Medium SupplementMix</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GENbox</td>
			<td>BioM&#233;rieux, Marcy l&#8217;Etoile, France</td>
			<td>96127</td>
			<td>2.5L</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Catalyst (AnaeroPack)</td>
			<td>MITSUBISHI GAS CHEMICAL&#160;COMPANY, INC.&#160;, Japan</td>
			<td>&#160;C-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anaerobic indicator</td>
			<td>BioM&#233;rieux, Marcy l&#8217;Etoile, France</td>
			<td>96118</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow cytometer</td>
			<td>Becton-Dickinson, USA</td>
			<td>FACSAria 2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BD FACSDiva Software</td>
			<td>Becton-Dickinson, USA</td>
			<td></td>
			<td>Version8.0.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sample tube</td>
			<td>Corning science, USA</td>
			<td>352054</td>
			<td>12*75mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS</td>
			<td>Hyclone, USA</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
	</tbody>
</table>

a flow cytometry-based assay for measuring mitochondrial membrane potential in cardiac myocytes after hypoxia/reoxygenation

Rettidig og effektiv reperfusion av okkludert koronar er den beste strategien for å redusere betennelsessykdommer som hjerteinfarkt størrelse hos pasienter med en ST-segmentet opphøyet hjerteinfarkt. Men kan reperfusion sådan føre videre cardiomyocyte død, et fenomen kjent som reperfusion skade. Åpningen av mitokondrie permeabiliteten overgangen pore (mPTP), med reduksjon av mitokondrie membranen potensielle (MMP) eller mitokondrie depolarization, er allment anerkjent som det siste trinnet av reperfusion skade og er ansvarlig for Mitokondrielt og cardiomyocyte død. JC-1 er et lipofile kationisk fargestoff som akkumuleres i mitokondrier avhengig av MMP. Jo høyere MMP er, jo mer JC-1 akkumuleres i mitokondrier. Den økende mengden JC-1 i mitokondrier kan gjenspeiles av et fluorescens utslipp skifte fra grønn (~ 530 nm) til rød (~ 590 nm). Derfor, reduksjon av rød/grønn fluorescens intensitet forholdet kan angi depolarization i mitokondrier. Her tar vi nytte av JC-1 å måle MMP eller åpningen av mPTP i menneskets hjerte myocytter etter hypoksi/reoxygenation, oppdaget av flowcytometri.

Før du utfører JC-1 analysen for å evaluere endringene av MMP, er det sterkt anbefalt at eksperimenter utføres for å bekrefte betingelsene er satt av forskerne. Som vist av flyt cytometri resultatene (figur 2), sammenlignet med normal gruppen, hypoksi/reoxygenation (H/R) betydelig indusert apoptose av HCMs (Annexin V + /PI±), som indikerer at vi hadde etablert en celle modell jeg / R (45.00 ± 2.13% vs. 11.50 ± 0,18% i normal gruppen p < 0,05)...

Watch this Scientific Journal Video about A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation at JoVE.com

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Her presenterer vi en protokoll som bruker JC-1 fargestoff for å vurdere mitokondrie membran potensialet i celler etter å bli utsatt for hypoksi/reoxygenation med eller uten en beskyttende agent.

En flyt cytometri-baserte analysen for å måle mitokondrie membran potensial i hjerte myocytter etter hypoksi/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

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The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

In vitro vurdering av myokardbeskyttelse etter hypotermi-prekondisjonering i en human hjerte-myocytter modell

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...