This work was supported by the Swiss National Science Foundation (310030_141070/1), Swiss Heart Foundation, and National Center of Competence in Research (NCCR-Kidney.CH) Switzerland. Yu Yi is supported by the Chinese Scholarship Council.

Hayvan çalışmaları Fribourg, İsviçre Veteriner Dairesi Etik Komitesi tarafından onaylandı. protokolü kurumda hayvan bakımı ve deney üzerinde yönergeleri takip eder. İzole Arter İnkübasyon için Set-up 1. Hazırlık <ol><li> 37 ° C&#39;ye kadar ısıtıldı ve bir karbon tankından% 95 O2 ve% 5 CO2 ile havalandırıldı edilebilir bir organ banyosu sistemi oluşturmak. </li><li> Aşağıdaki bileşimin konsantrasyonu ile gerektiği kadar Krebs-Ringer bikarbonat tamponu hazırlayın: (118 mM NaCI, 4.7 mM KCI, 2.5 mM CaCl2, 1.2 mM MgSO 4, 1.2 mM KH 2 PO 4, 25 mM NaHCO 3, 0.026 mM EDTA ve 11.1 mM D-glukoz). </li><li> Buz üzerinde stok tampon tutun ve% 95 O 2 ve% 5 CO 2 ile tampon havalandırır. </li><li> organ banyosu sistemi açın, 37 ° C&#39;de sıcaklığı ayarlamak her odasına hazır kullanıma tampon 5 ml ekleyinve% 95 O 2 ve% 5 CO 2 ile tampon havalandırmak tutun. </li><li> 30 dakika boyunca Krebs-Ringer tampon maddesi ile bir kez odaları yıkayın. </li><li> Her bölmeye 5 ml Krebs-Ringer tamponu ilave edin ve buharlaşmayı önlemek için bölme kapsamaktadır. </li></ol> Fare aorta 2. izolasyonu <ol><li> fareler kurban ila 150 mg / kg konsantrasyonunda intraperitonal% 0.9 NaCI içinde çözülmüştür pentobarbital enjekte edilir. </li><li> cerrahi gemide supinely fare Lay. </li><li> sanitizasyon ve nem amacıyla% 70 etanol ile göğüs bölgesinde kürk püskürtün. </li><li> kaburga çevresinde keserek açık göğüs boşluğu, kalp ve akciğerler maruz ve akciğerleri kaldırmak için. </li><li> Bir kağıt mendille hafifçe göğüs boşluğunda kan kaldırın. </li><li> Bir forseps ile yavaşça kalp kavrayın ve cerrahi makas ile aorta ve omurga arasındaki perivasküler yağ dokusunun kesim tarafından aorta ayırmak. </li><li> Hemen whol batırmakbuzla soğutulmuş (4 ° C) Krebs-Ringer tampon maddesi içinde e dokular. </li><li> Bir diseksiyon mikroskobu altında kalp ve aort arkı çıkarın ve tamponda torakal aort segmenti tutun. </li><li> cerrahi makas ve forsepsle bir mikroskop altında perivasküler, yapışan dokulardan aort teşrih. </li><li> Bir forseps ile aort uç kenarından tutun ve yavaşça bir 26 G × 1 / 2&quot; şırınga ile Krebs-Ringer tampon kızarma vasküler lümen kanı temizler. </li><li> 3 mm uzunluğundaki bölümler halinde temizlenmiş aort halkaları kesin. Not: endotel tabakasına zarar vermeyen bu aşamada dikkat ediniz. </li></ol> 3. DHE ve DAF-2DA Boyama <ol><li> 37 ° C&#39;de Krebs-Ringer tampon ile doldurulmuş bir organ banyosu odaları için forseps ile temizlenmiş aort segmentleri transfer% 95 O2 ve% 5 CO2 ile havalandırıldı. forseps ile aort halkaları Touch sadece adventisyal tarafı ve b kelepçe yok: NotLood gemiler. </li><li> 30 dakika boyunca organ banyosu odasında arterleri dengelenmesi. </li><li> nihai konsantrasyon 1 uM, organ banyosuna asetilkolin (Ach) ekleyin ve 10 dakika boyunca damar inkübe edin. </li><li> 1.5 ml mikrosantrifüj tüpü içinde önceden ısıtılmış Krebs-Ringer tampon maddesi ile (1.000 × stoktan seyreltilmiş, her boyanın 5 uM) DHE / DAF-2DA çözeltisi 1 ml hazırlayın. Işık maruz kalmasını önlemek için alüminyum folyo ile ependorf tüp sarın. </li><li> Stimülasyon sonrasında 37 ° C&#39;de, bir mikrosantrifüj tüpü içinde DHE / DAF-2DA çözeltisine organ banyosu arasında aortik halkaları transferi% 95 O2 ve% 5 CO2 ile havalandırılmıştır ve 30 dakika boyunca aort halkaları inkübe edin. Not: Bu adımda itibaren, karanlıkta hep aorta tutun. </li><li> Çamaşır yıkama için Krebs-Ringer tampon maddesi ile yeni mikrosantrifüj tüpüne aort halkaları aktarın ve 1 dakika içinde üç kere yıkama gibi tekrar edin. </li><li> 30 için sabitleme için% 4 paraformaldehid çözüm aort halkaları aktarınMin. </li><li> Hazırlama 1 mi 4 &#39;, fosfat tamponlu tuz (PBS), bir 1.5 ml mikrosantrifüj tüpü içinde tampon (1.000 × stoktan seyreltilmiş 300 nM), 6-diamidino-2-fenilindol (DAPI) çözeltisi. 3 dakika boyunca aorta counterstain. </li><li> 1 dakika boyunca yıkama için PBS tamponuyla yeni bir mikrosantrifüj tüpüne aorta aktarın. yıkamanın üç kez tekrarlayın. Not: Aort halkaları kelepçe veya endotel tabakası zarar vermemeye dikkat edin. </li></ol> Montaj 4. En Face <ol><li> slayt montaj orta bir damla ekleyin. </li><li> Haddelenmiş-up aorta düz yapabilir ve cam slayt aşağı bakacak şekilde endotel ile montaj ortamında yüz tr monte, mikroskop altında mikrocerrahi makas ile uzunlamasına aort halkaları kesin. ileri ve geri aorta hareket etmiyor. </li><li> kapak kayma ile slayt Kapak ve oje ile slayt mühür. </li><li> Işık koruma altına hava kurutulduktan sonra, korkunç görüntülenmesi için slaytlar kullanınctly veya saat içinde önümüzdeki birkaç gün için -20 ° C&#39;de saklayın. </li></ol> 5. Konfokal Mikroskopik Görüntüleme <ol><li> floresan sinyalleri algılamak için bir konfokal mikroskop kullanın. Makinenin açın ve büyütme, tarama hızı, çözünürlük, Z-yığını gibi görüntüleme ayarlarını optimize eder. <ol><li> Bu protokol için, bir 1024 × 1024 piksel, 200 Hz tarama hızı, çözünürlük ve 0.25 mikron Z adım boyutu kullanın. oysa 514 nm jeotermal gelen floresan heyecanlandırmak ve 605 nm emisyon de tespit, 488 nm argon lazer ile DAF-2DA gelen floresan heyecanlandırmak ve 515 nm&#39;de algılar. </li></ol></li><li> DAPI sinyali ultraviyole ışınları ile temizlenene kadar örnek üzerinde odaklanmak için 10X büyütme kullanın ve sonra 40X büyütme amacını ayarlayın. </li><li> Kontrol panelindeki Z konumunu ayarlayarak endotel tabakasının aralığını tanımlar. DAPI lekeli çekirdekleri sinyalleri endotel tabakasında oval veya yuvarlak noktalar vardır. </li><li> üst taramaendotel sinyal ve kayıt görüntülerin tam kalınlığı boyunca numunenin (lümen sınırında endotel tabakası). Her numune taramak için en az 3 farklı alanları seçin. </li></ol> Görüntüler 6. Analizi <ol><li> konfokal mikroskop tarafından taranan verileri açmak için yazılımı kullanın. analiz için alan başına art arda üç görüntüleri seçin ve numune en az 3 farklı alanlarda değerlendirmek. seçilen görüntüleri dışa ve JPEG dosyaları olarak kaydedebilirsiniz. </li><li> Görüntü işleme yazılımı ile boyanarak DAF-2DA jeotermal ve DAPI görüntüleri ölçmek. DAPI boyama gelen görüntü için, &#39;Eklentiler&#39; seçeneğini → DAPI pozitif çekirdeğin sayısını saymak için → &#39;Hücre Sayaç&#39; Analiz &#39;. <ol><li> DAF-2DA ve DHE boyaması gelen görüntüler için, sinyallerin yoğunluğunu analiz etmek → &#39;Tedbir&#39; &#39;Analiz&#39; seçin ve göreli sinyal yoğunluğu olarak &#39;Mean&#39; değerini alır. DAF-2DA oranına olarak mevcut sonra sonuçlarDAPI pozitif çekirdekleri veya DAPI için jeotermal enerjiden oranı. Her numune için, her görüntü ve alanın ortalama değerini alır. </li></ol></li><li> katlık değişime almak için kontrol grubunun ortalama her numunenin sonucunu bölün. İstatistiksel analiz Dunnett veya Bonferroni post-testi ile eşleştirilmemiş Student t testi ve ANOVA ile yapıldı. Ortalama ± SEM olarak veri verin. P &lt;0.05 14 anlamlı olarak ortalama değerleri arasındaki farkları göz önünde bulundurun. </li></ol>

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No conflicts of interest declared.

NO ya da O 2 tespiti .- floresan boyalar ile çoğu zaman dokuya cryosections 23 da kültürlü endotelyal hücrelerde pek çok çalışmada kullanılan ve. Burada yüz NO tespiti ve O 2 .-, nispeten basit ve sezgisel etkili sırasıyla DAF-2DA ve jeotermal, ile endotel tabakasında düzeyleri tr, yani sağlam bir yaşam kan damarları, bu yöntemi genişletilmiş. Bir arter medyada elastik lifler, özellikle cryosections aorta belirli NO ...

Vasküler endotelyal hücreler vazoaktif faktörlerin 1 serbest vasküler fonksiyonel ve yapısal bütünlüğünü korumak. Bu faktörler arasında, endotelyal NO sentazı (eNOS) ile L-arginin üretilen endotel kaynaklı nitrik oksit (NO), kardiyovasküler fizyoloji 2 en önemli ve en iyi karakterize edilen bir faktördür. NO dolayısıyla vasküler hastalık geliştirme 3 karşı koruma, endotel altı boşluğu içine trombosit agregasyonunu ve inflamatuar hücre yapışması ve infiltrasyonunu inhibe düz kas gevşemesi neden olur ve hücre çoğalmasını inhibe eder. Vb yaşlanma, hipertansiyon, diyabet, hiperlipidemi, aşağıdakileri içeren bir çok fizyolojik ve patolojik koşullar altında, karakterize endotel disfonksiyonu hiçbir biyolojik azalma ve O 2 .- üretimi mevcuttur ve ateroskleroz 2 patogenezi teşvik artmıştır. Son yıllarda yapılan çalışmalar eNOS ayrılması bir olduğunu göstermekÇeşitli yukarıda belirtilen koşullar 1,4 altında eNOS enzim O 2 ürettiği endotel disfonksiyonu, .- yerine NO için önemli bir mekanizma. Bu nedenle, endotelyal fonksiyon analizi, özellikle endotelyal NO üretimini ve O 2 .- nesil kardiyovasküler hastalıklar ve komplikasyonları hakkında deneysel araştırmalar için çok önemlidir. analiz eden ve biyolojik örneklerdeki NO üretimini ölçmek için geliştirilmiş bir çok yöntem yaklaşım vardır. Ve NO - 3 - nedeniyle kolaylıkla NO 2 oksitlenir NO derece kararsız olmaları 3 sn 6 arasında bir yarı ömürle, hiçbir doğrudan ölçülmesi çok zordur. / NO - 3 - NO 2 nedenle saptanması sıvısı örneklerinde hücrelere veya dokulara 5 salınan NO indeksi olarak kullanılmıştır. Prosedür, nispeten kolay olmakla birlikte, bu yöntem, ancak, EA/ HAYIR 3 - - çözeltide bulunan sily istikrarlı NO 2 yüksek arka etkilenir. NO siklik guanozin monofosfat (cGMP) 6 üretilmesi için çözünebilir guanilat siklazı uyaran için hücresel cGMP düzeyi de hiçbir serbest 7 tahmin etmek için belirlenmiştir. Yine, bu dolaylı tahmini ve C-tipi natriüretik peptidin (CNP) gibi endotel kaynaklı faktörler de parçacık guanilat siklazın 8 aktivasyonu yoluyla cGMP düzeylerini artırmak olabilir, çünkü spesifik olmayabilir. NO yan ürün 9, L-sitrulin, üretiminin ölçülmesi, bu nedenle, aynı zamanda, NO üretimini tahmin etmek için dolaylı bir yöntem olarak kullanılabilir bir şekilde L-sitrulin üretimi ile L-arginin ile elde edilir. Bu yöntemin en önemli dezavantajı radyoaktif olduğunu ve biyoaktif NO düzeylerini ölçmek olmadığını vardır, O 2 NO hızla inaktive olabilir bıraktığından beri .-; Öte yandan, L-sitrulin l- için geri dönüştürülebilir10 arginin. Bu kimyasal aydınlatma 11, elektron spin rezonansı, 12 veya elektrokimyasal porphyrinic sensör 13 gibi diğer kimyasal yöntemler bazı araştırmacılar tarafından kullanılmıştır. Bu yöntemler, genellikle tespit prosedürleri işletim kolay değildir ve özel ekipman gerektirir. Birçok çalışma ile veya endotel fonksiyonunu değerlendirmek ve dolaylı olarak endotel kökenli NO aracılı vasküler relaksasyon ölçmek endotel olmadan izole kan damarları ile organ banyosu deneyleri uygulamak söz aynı zamanda. çoğunlukla fizyolojik duruma yakın, ama olmasına rağmen Ancak, bu yöntem, kesinlikle söylemek NO işlevi ölçmez, oldukça bir üretim eNOS fonksiyonunun net etkilerini yansıtan genel olarak endotel aracılı vazomotor yanıtları değerlendiren diğer endotel kökenli gevşetici faktörleri ve endotel kaynaklı sözleşme faktörleri, O 2 .- üretimi, ve düz kas hücrelerinin de yanıtlarıBu faktörler. ENOS işlevi veya NO üretiminin özel bir analiz genellikle 3 gereklidir. Bizimki de dahil olmak üzere pek çok araştırma grupları YOK 14-19 hücre içi üretimini saptamak için floresan boya yöntemi kullanılan son yıllarda var. Bu yöntemde, hücre geçirgen floresan göstergesi diaminofluorescein-2 diasetat (DAF-2DA), in vitro ya da ex vivo hücre ve dokuları canlı kuru NO ve NOS fonksiyonu ölçmek için kullanılmıştır. prensibi canlı hücrelerde, DAF-2DA floresan olmayan 4,5-diaminofluorescein hücre içi esteraz suretiyle sitinin asetili alınır edilir bu o zaman ile reaksiyona sokularak DAF-2 triazol (DAF-2T) floresan dönüştürülmüştür (DAF-2) . DAF-2T floresan flöresanlı bir mikroskop veya flöresanlı konfokal mikroskop 14 altında gözlemlenebilir. Hücre içi floresan yoğunluğu dolayısıyla hücrelerde hücre içi NO üretimini veya sağlam kan vesse bir endotelyumuna yansıtırl. Böyle dihydroethidium (jeotermal) gibi belirli bir floresan boya ile birlikte, bir anda hücrelerde ya da kan damarlarında 14 hücre içi NO ve O 2 .- nesil değerlendirir. Benzer şekilde, DHE hücre geçirgen O 2 ile oksitlenir bileşik .- hücrelerin içinde ve daha sonra nükleik asitler ile intercalates floresan mikroskop veya floresan konfokal mikroskop ile kantitatif tespit edilebilen parlak kırmızı bir renk yayan oksidatif ürünü aynı zamanda. Bu, esas itibariyle, süperoksit radikalleri tespit hücreler tarafından iyi korunur ve hatta hafif tespit 20 tolere edilebilir DHE, O 2 .- biyolojik örneklerden saptanması için çok belirgin bir boyadır. Bu flöresan boyası yönteminin avantajlarından biri de tespit eder ve doğrudan bir oturma kan damarının sağlam endotel tabakası üzerinde cepheden NO ve / veya O 2 .- görselleştiren olmasıdır. Bu sayfada,Bu floresan boya NO algılamak için yöntem ve O 2 .- biz yüz NO tespiti ve O 2 tr için adapte olması tarif .- yüksek yağ-diyet (YYD) besleme tarafından uyarılan bir obezite fare modeli sağlam aorta içinde. Biz bu yöntemi başarıyla ve güvenilir NO ve O 2 .- düzeylerini ölçmek ve obezite taze izole edilmiş sağlam fare aorta endotel tabakasında eNOS (dis) fonksiyonunu değerlendirmek olabilir göstermektedir.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dihydroethidium (DHE)</td>
			<td>Invitrogen</td>
			<td>D 1168</td>
			<td>dissolve with DMSO to 5 mmol/L as 1000X stock, stored at -20&#176;C</td>
		</tr>
		<tr>
			<td>Diaminofluorescein-2 Diacetate (DAF-2 DA)</td>
			<td>Calbiochem</td>
			<td>251505</td>
			<td>dissolve with DMSO to 5 mmol/L as 1000X stock,stored at -20&#176;C</td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Invitrogen</td>
			<td>D 1306</td>
			<td>dissolve with water to 300 &#181;mol/L as 1000X stock,stored at 4&#176;C</td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>Vector labor. (reactolab)</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Name (N o-nitro-l-argininemethylester.HCl)</td>
			<td>Sigma-aldrich</td>
			<td>A5751</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Sigma-aldrich</td>
			<td>P3636</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Myograph System&#160;</td>
			<td>Danish Myo Technology A/S</td>
			<td>Model 610M</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope&#160;</td>
			<td>Leica&#160;</td>
			<td>DM6000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing software</td>
			<td>National Institute of Health(NIH)</td>
			<td>Image J&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors&#160;</td>
			<td>S&#38;T AG</td>
			<td>SDC-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical scissors&#160;</td>
			<td>F.S.T</td>
			<td>15000-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>S&#38;T AG</td>
			<td>JF-5</td>
			<td></td>
		</tr>
		<tr>
			<td>26G&#215;1/2&#698; syringe</td>
			<td>Becton Dickinson</td>
			<td>305501</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip round diam15mm</td>
			<td>vwr</td>
			<td>631-1579</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 1 mL</td>
			<td>vwr</td>
			<td>RFL-1200c&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 200 uL</td>
			<td>vwr&#160;</td>
			<td>613.0659</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes 1.5 mL</td>
			<td>Eppendorf&#160;</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylcholine chloride</td>
			<td>Sigma-aldrich</td>
			<td>A-6625</td>
			<td></td>
		</tr>
	</tbody>
</table>

en face detection of nitric oxide and superoxide in endothelial layer of intact arteries

endotelyal NO sentaz (eNOS) üretilen Endotel türetilmiş nitrik oksit (NO), kardiyovasküler fizyoloji en önemli vazoprotektif moleküllerden biridir. ENOS ayrılması süperoksit anyon NO biyoyararlanım ve artış azalmaya yol olarak işlevsiz eNOS gibi (O 2 .-) üretim ve sırayla kardiyovasküler hastalıklar teşvik etmektedir. Bu nedenle, endotel hücreleri NO ve O 2 .- seviyelerinin uygun ölçüm kardiyovasküler hastalıklar ve komplikasyonları hakkında araştırma için hayati önem taşıyor. Çünkü NO ve O 2 .- son derece kararsız doğasından ötürü, herhangi bir önlem ve O 2 .- doğrudan kan damarına zordur. Birçok yöntem HAYIR ölçmek ve O 2 .- üretim için geliştirilmiştir. Ancak, her iki hassas olmayan ya da spesifik olmayan ya da teknik olarak zor ve özel ekipman gerektirmektedir. Burada bir adaptasyon tarifYüz eş zamanlı olarak saptanması ve hücre içi NO ve O 2 görselleştirme tr .- kaynaklı bir obezite fare modeli sağlam aorta sırasıyla, hücre geçirgen diaminofluorescein-2 diasetat (DAF-2DA) ve dihydroethidium (DHE) kullanarak floresan boya yöntemi yüksek yağ-diyet beslenme ile. Kontrol yalın fare ile karşılaştırıldığında Biz obezite fare taze izole edilmiş sağlam aorta 2 .- seviyeleri hücre içi NO azalmış ve O zengin oldukları olabilir. Bu yöntem, doğrudan algılama ve görselleştirme NO ve O 2 için kolay bir teknik, .- sağlam kan damarlarında ve yaygın (fizyo) patolojik koşullarda, endotelyal (dis) fonksiyonunun incelenmesi için uygulanabilir olduğunu göstermektedir.

Obezite iskemik koroner kalp hastalığı için önemli bir risk faktörüdür ve ilişkili NO biyoyararlanımını, aterosklerotik damar hastalığı 21 bir işaretidir endotel azalır. eNOS-ayrılması 22, ateroskleroz ve obezite 14 yaşlanma da dahil olmak üzere bir çok fizyolojik ve patolojik koşullar altında endotel disfonksiyonu için önemli bir mekanizma olduğu gösterilmiştir. Bu nedenle, burada temsili NO sonucunu ve O 2 .-...

Watch this Scientific Journal Video about En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries at JoVE.com

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Bozulmamış Arterlerin endotel katmanı Nitrik Oksit ve Süperoksit En Yüz Algılama

Bu yazıda floresan boya tr yüz eşzamanlı algılama ve hücre içi nitrik oksit görselleştirme (NO) ve süperoksit anyon (O 2 diaminofluorescein-2 diasetat (DAF-2DA) ve dihydroethidium (DHE) kullanarak adapte nispeten kolay bir yöntem tarif .- ) sırasıyla, taze bir obezite fare modelinin sağlam aorta izole edilmiştir.

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

en-face-detection-nitric-oxide-superoxide-endothelial-layer-intact

Research

JoVE Journal

Biology

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

9.5K Görüntüleme.  University of Fribourg.  Bu yazıda floresan boya tr yüz eşzamanlı algılama ve hücre içi nitrik oksit görselleştirme (NO) ve süperoksit anyon (O 2 diaminofluorescein-2 diasetat (DAF-2DA) ve dihydroethidium (DHE) kullanarak adapte nispeten kolay bir yöntem tarif .- ) sırasıyla, taze bir obezite fare modelinin sağlam aorta izole edilmiştir. 

Video: En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

ELISA yöntemi ile yerli bozulmamış nükleozom Post-translasyonel modifikasyonlar Algılama

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Biyoloji

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Nitrik Oksit ve hücreler arası Elektron Paramanyetik Rezonans Spektroskopisi ile süperoksit Radikal Anyon tespiti Spin Tuzakları kullanarak

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Fare Direnç arterlerden mikrovasküler endotel Tüpler İzolasyonu

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Tek Mitokondriyal süperoksit Bozulmamış Kalp Flaşlar veya konfokal Görüntüleme<em&gt; In Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Zebra balığı Embriyolar Oksidatif Stres Analizi

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Hücre içi Ca İki foton Görüntüleme<sup&gt; 2+</sup&gt; Bir İzole Sıçan Aort Kullanım ve Endotel Nitrik Oksit Üretimi ve düz kas hücreleri

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Gelişim ve karakterizasyonu<em&gt; İn Vitro</em&gt; Mikrodamar Ağ ve Endotel kantitatif ölçümleri [Ca<sup&gt; 2+</sup&gt;]<sub&gt; I</sub&gt; Ve Nitrik Oksit Üretimi

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Montaj ve &#39;Kayma Rings&#39; Uygulama: Orbital tek yönlü ve Periyodik Akışkan Akışının ve Kayma Stres için Yeni Bir Endotel Modeli

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

Kemilüminesans Yöntemi kullanılarak biyolojik Malzemeleri, Nitrik Oksit Yolu&#39;ndaki nitrit ve nitrat, Metabolitler Ölçme

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

En Face Preparation of Mouse Blood Vessels

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the three-dimensional tissue morphology is from such sections. In addition, the sections of tissues examined may not contain the region within the tissue that is necessary for the purpose of the ongoing study. This latter limitation hinders histopathological studies of blood vessels since vascular lesions develop in a focalized manner. This requires a method that enables us to survey a wide area of the blood vessel wall, from its surface to deeper regions. A whole mount en face preparation of blood vessels fulfills this requirement. In this article, we will demonstrate how to make en face preparations of the mouse aorta and carotid artery and to immunofluorescently stain them for confocal microscopy and other types of fluorescence-based imaging.

En Face Preparation of Mouse Blood Vessels

A procedure for making en face preparations of the mouse carotid artery and aorta is described. Such preparations, when immunofluorescently stained with specific antibodies, enable us to study localization of proteins and identification of cell types within the entire vascular wall by confocal microscopy.

Fare Kan Gemilerinin Yüz Yüzey Hazırlığı

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the ...