Name: isolation and primary culture of mouse aortic endothelial cells
Uploaded: 2016-12-19T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

This study is supported by American Heart Association Scientist Development Grant 13SDG16930098 and the National Science Foundation of China Youth Award 81300240 (PI: Wang). We thank Roberto Mendez from Wayne State University for assisting in the preparation of the manuscript.

1. Isolatie van Aorta van Muizen Al de hier beschreven procedures werden goedgekeurd door de Institutional Animal Care en gebruik Comite van Wayne State University. <ol><li> Zet de muis in de inductie kamer van het anesthesieapparaat. Stel de isofluraan stroom naar 4% samen met 25% verse lucht en 75% O2. Verdoven van de muis door inademing van isofluraan (4% voor inductie, ± 1,0% voor het onderhoud) in combinatie met 25% lucht fris en 75% O 2 via inductie ka...

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Deze studie toont een eenvoudige methode om te isoleren en cultuur endotheliale cellen van een muis aorta zonder speciale apparatuur. De immunofluorescentie kleuring gaf aan dat de meerderheid van de cellen endotheliale cellen na de tweede doorgang. Er wordt gesuggereerd dat cellen in tweede tot derde doorgang geschikt voor in vitro en in vivo experimenten endothele studie biologie. De Key Notes from the Present Protocol Er zijn...

Het vasculaire endotheel is niet alleen een barrièrelaag die bloed en weefsel scheidt, wordt het beschouwd als een grote endocriene klier die zich uitstrekt over de gehele vaatstelsel met een oppervlakte van 400 vierkante meter 1. Het welzijn van het endotheel essentieel vasculaire homeostase. Disfunctionele endotheel participeert in diverse cardiovasculaire aandoeningen, zoals atherosclerose, vasculitis en ischemie / reperfusie letsel, etc. 2-4. Tot op heden worden de specifieke cellulaire en moleculaire mechanismen die betrokken zijn bij deze ziekte instellingen niet goed begrepen als gevolg van de diffuse anatomische aard van het endotheel. De muis is een belangrijk model voor het onderzoek, omdat genetische manipulatie technieken zijn meer ontwikkeld in muizen dan in elke andere zoogdiersoorten. Echter de isolatie van primaire muis endotheelcellen als bijzonder moeilijk omdat de geringe omvang van de aorta maakt enzymatic vertering van endotheel onpraktisch. Verklaarden sommige procedures voor het isoleren en zuiveren EC vereist 5-7. Het doel van dit protocol is een eenvoudige methode te isoleren en te expanderen endotheliale cellen van de muis aorta zonder speciale apparatuur. In dit protocol wordt de vers geïsoleerde aorta in kleine segmenten en gezaaid op een matrijs met het endothelium naar beneden om voor endotheliale kiemen. Na segmenten worden verwijderd, worden endotheelcellen uitgebreid in-endotheel voorkeur medium en zijn klaar voor experimenten na twee of drie passages. De voordelen van de beschreven werkwijze zijn de volgende: 1) aanzienlijk hoge aantallen endotheelcellen worden geoogst van een aorta; 2) levensvatbaarheid van de cellen is goed bewaard gebleven; en 3) geen speciale uitrusting of techniek nodig. Het een effectief middel van het identificeren van specifieke cellulaire en moleculaire mechanismen endotheelcellen pathofysiologie. Voor degenen die geïnteresseerd zijn in het bestuderen van zijn primary gekweekte endotheelcellen van ofwel gen knock-out muizen, gen knock-in muizen of muizen ziekte model, dit protocol is erg handig en makkelijk om te oefenen.

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			<td>4- or 6-week-old mice (Jackson Laboratory, #000664).</td>
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			<td>Sterile 1X phosphate-buffered saline (PBS, Gibco, #10010-023).</td>
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			<td>Sterile 1X PBS containing 1,000 U/ml of heparin (Sigma Aldrich, H3149).</td>
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			<td>Endothelial cell growth medium (Dulbeccos&#8217; Modified Eagle&#8217;s Medium[DMEM] with 25mM HEPES[Gibco, #12320-032 ], supplemented with 100&#956;g/ml endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma,#2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000 U/ml heparin [Sigma Aldrich, H3149], 10,000U/ml penicillin and 10mg/ml streptomycin [Gibco, #15140-122]).</td>
		</tr>
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			<td>Growth factor reduced matrix (BD Biosciences, #356231).</td>
		</tr>
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			<td>Neutral proteinase (Dispase, 1U/ml, Fisher Scientific #CB-40235) and D-Val medium (D-Valine, 0.034g/L[Sigma, #1255], in Dulbeccos&#8217; Modified Eagle&#8217;s Medium, low glucose[Gibco, #12320-032 ]).&#160;</td>
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			<td>Gelatin (100&#8209;200 &#956;g/cm2, Sigma, #G1393).</td>
		</tr>
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			<td>1,1`-dioctadecyl-3,3,3`,3`- tetramethylindo- carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL, Life Technologies, #L3484),&#160; FITC-labeled Ulex europeus agglutinin (Ulex-Lectin, Sigma, #L9006),&#160; Anti-mouse CD31-FITC conjugated antibody (BD Biosciences, # 553372).</td>
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			<td>Anti-mouse vascular endothelial growth factor receptor 2 antibody (Cell signaling, #9698), anti-mouse endothelial nitric oxide synthase (Abcam, #ab5589), anti-mouse vascular endothelium-cadherin (Abcam, #33168), anti-mouse calponin (Abcam, #700), FITC-conjugated anti-rabbit IgG (Sigma, #F6005)</td>
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			<td>One ml syringe fitted with 25-G needle (Fisher Scientific, #50-900-04222).&#160;</td>
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			<td>100mm Peri dishes (Fisher Scientific,&#160; #07-202-516).</td>
		</tr>
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			<td>Six-well cell culture plates (Fisher Scientific, #08-772-1B).</td>
		</tr>
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			<td>T12.5 cultuer flask (Fisher Scientific, #50-202-076)</td>
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			<td>Scissors, forceps, microdissection scissors and forceps, Scalpel blade (Fine Science Tools, Inc)</td>
		</tr>
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			<td>Anesthesia machine with isoflurane (Webster Veterianary Supply, #07-806-3204), heating lamp</td>
		</tr>
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			<td>Centrifuge machine.</td>
		</tr>
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			<td>Inverted phase-contrast microscope.</td>
		</tr>
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			<td>inverted fluorescence microscope.</td>
		</tr>
	</tbody>
</table>

isolation and primary culture of mouse aortic endothelial cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

Endotheelcellen Kiemen Spontane endotheelcellen kiemen gestart vanuit een muis aorta segment. De muisaorta segment liet men groeien op een groeifactor gereduceerde matrix dan in endotheliale celgroei medium voor 4 dagen. De endotheelcellen kiemen verschijnt meestal in 2-4 dagen. Microfoto&#39;s werden genomen op dag 4 (Figuur 1). Zoals getoond in de foto&#39;s, talrijke endotheelcellen migreren van het segment. De nieuw gevormde spruiten blijven uitstrekken ...

Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolatie en Primaire Cultuur van de muis endotheelcellen

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

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