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

Bu çalışmada ulusal anahtar araştırma ve geliştirme programı of China (No. 2017YFC1700503), gelen hibe tarafından desteklenen Ulusal temel araştırma programı (973 programı), Çin (No.2012CB518602), Ulusal Doğa Bilimleri Foundation of China (No. 81370223 ve No 81573957) ve öngörüyor yenilikçi Araştırma Vakfı Pekin Birliği Medical College (2016-1002-01-02).

1. hazırlanması reaktifler ve çözümleri
<ol><li>İnsan kardiyak miyositler (HCMs) tam orta Myocyte büyüme orta 500 ml üretici yönergelerine göre 25 mL Myocyte büyüme orta ek karışımı ekleyerek hazırlayın. 4 ° C ve kullanmadan önce 37 ° c sıcak mağazasında.</li>
	<li>Tongxinluo (TXL) çözüm hazırlamak TXL süper ince toz serum/glikoz-Alerjik içinde eriterek tarafından Dulbecco'nın kartal Orta (DMEM) değiştiren ve TXL konsantrasyon 400 µg/ml DMEM (daha fazla bilgi için bkz:<sup class="xr...

<ol>
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Burada, biz H/R. tespit için Dizayn Merkezi-1 tahlil tarafından maruz kalma sonra MMP hücre değerlendirmek için Dizayn Merkezi-1 boya kullanan bir iletişim kuralı mevcut, hücre MMP mitokondrial boyut, şekil ve tek bileşenli floresans etkileyebilir yoğunluğu gibi faktörlerin bağımsız 14sinyalleri. Sonuç olarak, JC-1 tahlil sonuçlarını görece güvenilirdir. Ayrıca, uygun ve zaman kazandıran JC-1 tahlil gerçekleştirmek için. Bu tahlil malzemeleri ve Kimyasalları için düş...

Koroner kalp hastalığı ölüm dünya çapında önde gelen nedenidir. İskemik yaralanma azaltmak ve ST-segment olan hastalarda enfarktüsü boyutu sınırlama miyokard infarktüsü yükseltilmiş için seçim zamanında ve etkili miyokardiyal reperfüzyon birincil perkütan koroner girişim (PCI)1üzerinden tedavisidir, 2. Ancak, reperfüzyon son enfarktüsü boyutu3ilâ yüzde 30'hesap ek hasara neden olur. Evrensel mitokondrial geçirgenliği geçiş gözenek (mPTP) sadece iskemi/reperfüzyon sırasında mitokondrial hasar ve hücre ölümü Central'da değil kabul edilmektedir (ı / R), ama aynı zamanda4 sinyal kardiyoprotektif yakınsak bir hedeftir , 5. mPTP açılış iç mitokondri zar potansiyel (MMP)4depolarizasyon hakkında getirmek gibi tespit ettik 5 ' 5 ', kullanarak mPTP açılış 6, 6 '-Pekala-1, 1, 3, 3 '-tetraethyl-imidacarbocyanineiodide (JC-1) tahlil.
JC-1 tahlil hem nitel hem de nicel bir cytofluorimetric yöntemidir ve bir tek mitokondri6düzeyinde MMP analiz ederek daha da doğrulandı. JC-1 kırmızı turuncu renkli emisyon (590 ± 17.5 nm) matris ile normal MMP mitokondri için verimli bir araya getirilmiş biçimi olarak, var; MMP kaybı ile Dizayn Merkezi-1 salma 530 ± 15 Nm ile yeşil floresans verimleri monomeric forma dönüşür. Bu nedenle, kırmızı/yeşil floresan yoğunluğu oranında bir düşüş MMP azalma iskemi/reperfüzyon gibi koşulları göstergesi olabilir (ben / R).
JC-1 ek olarak, MMP da rodamine 123 ve 3, 3'dihexyloxadicarbocyanine iyodür [DiOC6(3)] gibi membran geçirgen lipofilik katyonlar ile çalışılmıştır. Ancak, bu iki problar için karşılaştırıldığında, JC-1 MMP analiz etmek için daha güvenilirdir. Rodamine 123 (özellikle de modu7,8Şoklama) nispeten yoksul duyarlılık ve zavallı özgüllük vardır. Rodamine 123 vardiyada bazen gözlemlemek/algılamak araştırmacılar veya ekipmanlar için zor kadar küçük. Ayrıca, tek bir hücrede farklı mitokondri bağlayıcı siteleri rodamine 123 için vardır ve bu kadar farklı floresan emisyonları9çok olabilir. MMP algılamak için tavsiye edilmez DiOC6(3) hassas plazma zarı10depolarizasyon için tepki olarak da.
Bu nedenle, burada biz Dizayn Merkezi-1 tahlil HCMs MMP hipoksi/reoxygenation veya koruyucu bir ajan olmadan maruz sonra değerlendirmek için kullanın.

<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

Zamanında ve etkin reperfüzyon tehlikesi koroner arter ST segment olan hastalarda miyokard enfarktüsü boyutunu azaltmak için en iyi strateji miyokard infarktüsü yükseltilmiş olduğunu. Ancak, reperfüzyon başına daha fazla cardiomyocyte ölüm, reperfüzyon hasarı bilinen bir olay neden olabilir. Mitokondri zar potansiyel (MMP) azalma veya mitokondrial depolarizasyon, mitokondrial geçirgenliği geçiş gözenek (mPTP), açılış evrensel reperfüzyon hasarı son adımı olarak kabul ve sorumludur mitokondri ve cardiomyocyte ölüm. JC-1 MMP değerine bağlı olarak mitokondri içinde biriken bir lipofilik katyonik boya var. Yüksek MMP, daha Dizayn Merkezi-1 mitokondri içinde birikir. Mitokondri artan miktarlarda JC-1 yeşil floresans emisyon vardiya tarafından yansıması olabilir (~ 530 nm) kırmızı (~ 590 nm). Bu nedenle, kırmızı/yeşil floresan yoğunluğu oranı azaltma mitokondri depolarizasyon belirtebilirsiniz. Biz MMP ölçmek için Dizayn Merkezi-1 avantajı veya hipoksi/reoxygenation akış sitometresi tarafından tespit edildi sonra insan kardiyak miyositler mPTP açılış al.

MMP değişiklikleri değerlendirmek için Dizayn Merkezi-1 tahlil yapmadan önce deneyler koşulları başarıyla onaylamak için araştırmacılar tarafından ayarla yürütülen bu önerilir. Normal grup ile karşılaştırıldığında akış sitometresi sonuçları (resim 2), tarafından gösterildiği gibi hipoksi/reoxygenation (H/R) önemli ölçüde HCMs (Annexin V + /PI±), apoptozis indüklenen hücre tabanlı modeli ben kurmuş gösteren / R (45.00...

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

Burada, hipoksi/reoxygenation veya koruyucu bir ajan olmadan maruz sonra hücreleri mitokondrial membran potansiyelini değerlendirmek için Dizayn Merkezi-1 boya kullanan bir iletişim kuralı mevcut.

Kardiyak miyositler mitokondrial membran potansiyeli hipoksi/Reoxygenation sonra ölçmek için akış sitometresi tabanlı tahlil

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Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Kalp Damar Cerrahisi sonra serum Örnekleri etkilenerek Sağlıklı Gönüllüler ve Bunların Göçmen Potansiyelden Endotel Ata Hücreleri İzolasyon

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Burun membran Transepithelial potansiyel farkının (NPD) standart ölçüm

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) ...

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

HiPSC elde edilen kalp kası yamalar implantasyonu eskiden şiling şimdi domuz modelinde miyokardiyal yaralanma sonra

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

Isolation of Atrial Myocytes from Adult Mice

Yetişkin fareler atriyal Miyoksit izolasyonu

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

İnsan Kan Akışını Taklit Eden Akış Döngüsü Modelinde Kanla Temas Eden İmplantların Hemokomuuyum Testi

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

İnsan Kardiyak Miyosit Modelinde Hipotermi-Önkoşullama Sonrası Miyokard Korumasının İn vitro Değerlendirmesi

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