Biz görüntüleme çalışmaları ile yardım için Dr. William Cashdollar ve Wisconsin Kan Araştırma Enstitüsü&#39;nde Kuzeybatı Karşılıklı Vakfı Görüntüleme Merkezi&#39;ni teşekkür ederiz. Biz de bu yazının ve yararlı bir tartışma eleştirel okuma Dr Daria Ilatovskaya teşekkür ederim. Bu çalışma (AS) Sağlık Hibeler HL108880 National Institutes ve (AMG için) DP2-OD008396 tarafından desteklenmiştir.

Aşağıda açıklanan deney prosedürleri Wisconsin Tıp Koleji Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmış ve Laboratuvar Hayvanları Bakım ve Kullanım Sağlık Rehberi National Institutes uyarınca idi. Sıçan Aort 1. İzolasyonu <ol><li> Izofluran (% 5 indüksiyon,% 1.5 2.5 bakımı) veya başka bir IACUC ile sıçan uyutmak yöntemi onayladı. </li><li> Bir yatar pozisyonda sıçan yerleştirin. Karın organları açığa çıkarmak amacıyla, deri ve deri altı karın dokusu sayesinde küçük bir ventral orta hat kesi yapmak. Şekil 1 &#39;de gösterildiği gibi kullanılması gazlı bez yavaşça aort ve vena cava açığa çıkarmak için bağırsak saptırır. </li><li> Kısaca, bağ dokusu ve vena kava gelen aort teşrih künt. Böbrek mümkün olduğunca yakın aort kapalı kravat, dikiş kullanma. Aort 2 cm ve 15 ml konik tu yerleştirin - yaklaşık 1 teşrihNormal fizyolojik tuz çözeltisi içeren olabilir (PSS; mM olarak: 140 NaCl, 1.2 MgCl2, 2 CaCl2, 4.5 KCI, 6 Glikoz, 10 HEPES), buz üzerinde. </li><li> Aort izole edildikten sonra, onaylanmış IACUC protokollere göre hayvan euthanize. Olmayan tüm yaşam bitiminde prosedürleri hayvanın insani ölümünü sağlamak için pnömotoraks uyaran torakotomi ile derinden anestezi hayvanları euthanize. 3 </li><li> Bir diseksiyon mikroskop kullanarak, forseps ince uçlu ve microscissors aort kapalı yağ ve bağ dokusu teşrih. Aort temiz kez uzunlamasına aort kesip daha sonra buz üzerinde PSS yerleştirin. </li></ol> 2. Boya Yükleme ve Kuluçka <ol><li> Pipet alüminyum folyo ile kaplanmış olan tek tek 500 ul tüplere, DMSO-bazlı çözelti içinde temin edilmektedir 1mM Fluo-04:00 boya, 7 ul. Zamanla boya özelliklerini korumak için -20 ˚C de dondurucuda saklayın. Not: Bu yüzden aliquotedmel çözümler, en az altı ay stabil olmalıdır. </li><li> Kullanımdan önce, boya çözeltileri açmadan önce oda sıcaklığına ısınmaya bırakın. Kullanımdan hemen önce çalışan çözümleri hazırlayın. Daha sonra kullanılmak üzere sulandırılmış reaktifi tutmayın. </li><li> Bir kalsiyum içeren PSS 450 ul DMSO çözeltisi içinde çözülmüştür hazır 1 mM Fluo-04:00 boya 7 ul ekleyin. Asetoksimetil (AM) esterleri dağıtmak yardımcı ve kalsiyum boya yükleme iyileştirmek için yükleme kokteyli olmayan DMSO bazlı% 10 Pluronic Asit çözeltisi 40 ul ekleyin. </li><li> 1 saat boyunca (as 2.3 açıklanmıştır) yükleme kokteyli içeren 500 ul tüpler içine disseke aorta yerleştirin. Oda sıcaklığında bir döner çalkalayıcı üzerinde folyo ve yer ile tüm tüpler örtün. </li><li> Damar NO üretimini izlemek için DAF-FM diasetat boya kullanın. 450 ul PSS, 40 ul pluronik asit ve 10 mM DAF-FM diasetat 5 ul ihtiva eden bir çözelti içinde aorta inkübe edin. (2.3 açıklanmıştır) kalsiyum kuluçka protokolüne benzer, aort yerleştirinoda sıcaklığında Sonraki 30 dakika, ardından 4 ° C&#39;de 30 dakika için bir döner sallayıcı üzerinde folyo ve yerine kaplı NO boya çözeltisini ihtiva eden bir 500 ul bir tüp içinde. </li><li> NO üretimi ölçüldüğünde, Şekil 4B&#39;de gösterildiği gibi, bir kontrol olarak NO üretimini engellemek için, endotel NO sentaz engelleyici, L-NAME kullanın. Bu işlem için, (oda sıcaklığında) inkübasyon son 30 dakika süreyle (2.5 de anlatıldığı gibi) kokteyl 100 nM L-NAME ekleyin. </li></ol> 3. Lazer Tarama İki foton Mikroskopi Protokolü <ol><li> Dışı boya kaldırmak için temiz PSS 3 kez - 1 saat inkübasyondan sonra, aorta 2 yıkayın. </li><li> Normal PSS veya Ca 2 + -ücretsiz tampon (deney protokolünde bağlıdır) ya içeren bir silikon kaplı çanak aort aktarın. Silikon ile temas adventisya aşağı silikon kaplama üzerine aorta Pin (yani çanak açılış maruz kalan endotel lümen) μ şeklinde işaretçilerine kullanarak. Alternatif olarak, bir dilim Anchor kullanınızgara veya tutkal dokusunu düzeltmek için. Not: stres ve olası mekanik eserler, transferi azaltmak ve Ca 2+ -ücretsiz çözüm aort düzeltmek ve 5 beklemek için - deney başlamadan önce 10 dakika. </li><li> Peristaltik bir pompa ya da ilaçların, otomatik veya manuel uygulama ya da hücre-dışı çözelti değişen silikon kaplı plakaya bir şırınga bağlayın. </li><li> Dik iki foton mikroskop burun parçasının altında çanak yerleştirin ve yüklemek ve numune üzerinde 25 × (NA 1.05 ve çalışma mesafesi 2 mm) su daldırma objektif lens yerleştirin. Not: Bu özel bir mercek mevcut değilse normal bir amaç ile karşılaştırıldığında önemli ölçüde sinyal çözünürlüğünü artırabilir, çünkü özellikle iki foton görüntüleme için üretilmiş bir objektif kullanın. </li><li> Yazılımını kullanarak iki foton lazer etkinleştirin (lazer pompa ve açmak için başlar). Dinle 820 nm lazer uyarım. </li><li> Epifloresans kullanarak, aort bulmak ve üzerine objektif odakKaba ayarı kullanarak endotel yüzey. </li><li> Uyarma lazer modu kilitli olmayan descanned dedektörler ilgileniyoruz ve beklenen emisyon spektrumları uygun emisyon filtreleri yüklü olmasını sağlamak, iki foton lazer tarama moduna mikroskop Anahtarı. Not: Burada FV10-MRL / R (495 nM 540) kullanılmıştır. </li><li> Yaklaşık% 5, lazer gücünü kullanarak canlı taramayı başlatmak ve ince endotel hücrelerini görselleştirmek için objektif odak. Not: Normal PSS inkübe zaman endotel hücreleri güçlü bir floresan sinyal sergileyecek. Gemi, kalsiyum içermeyen tampon maddesi içinde inkübe edilir, bu fark daha zayıf olacaktır. endotelial hücreler açılır aort üst ve düz kas hücreleri, sadece endotel hücreleri (bakınız Şekil 2) aşağıda görülebilir mevcut olacaktır. Kan damarları düz ne de düz kas hücreleri ve endotel hücrelerinin her ikisi de mevcut yerler olacak ne olduğundan. &lt;/ Li&gt; <li> Hücreler odak olduğunda, mikroskop yazılım programı hızlı tarama modunda sıralı görüntüleri (raster tarama, 1.1 sn kare toplama frekansı ile 512 x 512 piksel pencere) toplamak için. Flu-4:00 floresan paralel olarak, damar daralması değişiklikleri algılamak ve daha iyi deneysel koşullar görselleştirmek için ışıklı görüntüleri bulaşan toplamak. </li><li> Bazal sinyal görüntüleri toplandıktan sonra, harici bir çözelti içinde Ca2 + iyonu konsantrasyonu değiştirmek ya da yavaş yavaş peristaltik pompa görüntüleme sırasında kullanarak farmakolojik uyaran maddeler uygulanır. Yüklenen hücrelerin içindeki floresan sinyal artışı dikkate alınmalıdır. Not: Endotel hücreler, akış değişikliklere bu nedenle Ca 2+ veya hiçbir sinyal farmakolojik aktivatörleri uygulanmasından önce, düşük laminer uygulamaları ve araç test kullanılması önerilir yanıt. Fluo-4 (Fluo-4 i için ayrışma sabiti yüklü gemiler nM konsantrasyon değerlerine hücre içi kalsiyum yeniden hesaplamak içins 345 nM), flüoresan yoğunluğu, taban çizgisinde ve Genişleme ve MnCI2 eklenmesinden sonra kaydedilebilir. 4 </li></ol> 4. Görüntü İşleme ve Hesaplamalar <ol><li> Download ve Fiji görüntü işleme paketini yükleyin. </li><li> Fiji görüntü dosyasını açın. İstendiğinde dosyasını içe üzerine, kanallar (iletilen ışık ve florasan sinyali) bölün. Veri analizi için sadece floresan sinyal kullanın. </li><li> Fiji programı kapsamında, analiz sekmesini tıklayın ve araçları seçeneğine gidin. Araçlar seçeneği altında, ROI yöneticisi diyor sekmeyi bulmak ve üzerine tıklayın; Yeni bir pencere açılacaktır. </li><li> Bundan sonra, analiz sekmesi altında ayarlanan ölçümlerine tıklayın. Özel ilgi ölçümleri seçin. Kalsiyum geçici ölçümler için ortalama gri değer seçeneğini kullanın. </li><li> Dairesel iz aracı ile, ilgi alanları (ROI) bölgelerini izlemek ve her R ROI yöneticisi ekranda &quot;eklemek&quot; butonuna tıklayınız başlarOI. Bir kez ilgi tüm hücreleri ROI yöneticisi penceresinde, &quot;daha&quot; sekmesi altında Çoklu Tedbir seçin takip edilmiştir. Bir elektronik tabloya bu ölçümlerin hepsi doğrudan ölçümleri kaydetmek veya kopyalayıp yapıştırın ya unutmayın. </li><li> Açık * .txt dosyası veya Excel Fiji ve yeni bir çalışma sayfasına Kökeni numaraları aktarın. Not: Alternatif olarak, daha fazla analiz için Sigma Plot veya Prism gibi herhangi bir program kullanın. </li><li> Origin programı kapsamında, tüm geçici tepkilerin bir bakış gözlemlemek için Line + Sembol menüsü → Plot kullanın. Tepkisiz hücreleri kaldırın. </li><li> Zaman ölçeği (X ekseni) iz numarasını yeniden hesaplayın, Sütun → Set Sütun Değerlerini kullanın ve (bizim durumumuzda 1,1sn olarak) resim koleksiyonu frekansına iz numarası sütunu çarpın. </li><li> Mean (Y ekseni) ve Standart Hata izleme hesaplamak için seçilen tüm kayıtlar için Satır Analysis → İstatistikleri kullanın. Hücrelerin mevcut grup Line + Sembol → Konu için son Grafik çizilir. </li><li> meaolarak takip n kalsiyum geçici hesaplama açıklanmıştır. <ol><li> Kökeni program dahilinde toplam kalsiyum salınımı için, daha sonra, Grafik tıklayın menüsü Analizi → Matematik kullanın → entegre edin. Eğri altında kalan alan toplam ayrılmaz Sonucu giriş görünür. </li><li> (X ekseni geçici yanıt sonuna kadar maksimum aralığı seçin) Analiz → Doğrusal olmayan Curve Fit → Seçiniz Veri Seti: Geçici kinetik için Graph üzerine tıklayın, ardından menüyü kullanabilirsiniz. Bitti → Üstel Çürüme 1 → Seç Fonksiyonu → Montaj başlatın. Üstel uydurma Grafik penceresinde görüntülenir ve Giriş Sonuç: Not. Elde edilen veriler (değerler) yayımlanan kalsiyum toplam miktarını temsil ve kanalların kinetik geçici yanıt aracılık. </li></ol></li></ol>

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Yazarlar ifşa hiçbir şey yok.

Deneysel Bakış. Daha iyi damar fizyolojisi HAYIR kalsiyum katkısını anlamak ve için, yeni bir yöntem ölçmek için geliştirilen [Ca 2 +] i ve NO düz kas ve izole bozulmamış aorta endotel hücreleri içinde. 1) Mekanik izolasyon ve hazırlık (enzimatik olmayan sindirim) Geminin: Birlikte, bu protokol bu kritik adımlardan oluşur. Optimum fizyolojik kayıtları elde etmek mümkün olduğunca sağlıklı ve sağlam dokuyu tutmak önemlidir. Pluronik asit ile 2) kalsiyum etiketle...

Kalsiyum, endotel ve düz kas hücreleri gibi vasküler hücreler içinde temel bir ikinci mesajcı olan. Bu vasküler kasılma için birincil uyaran ve endotel içinde YOK nesil yoluyla etkileri de dahil olmak üzere, vasküler dilatasyon önemli bir rol oynamaktadır. Nedeniyle görüntüleme teknolojilerinin sınırlamaları nedeniyle, sağlam geminin içinde kalsiyum işleme gözlemlemek neredeyse imkansız olmuştur. İki foton görüntüleme sistemleri ve yeni kalsiyum yaratılması veya HAYIR etiketleme boyalarının geliştirilmesi, kalsiyum dinamikleri ve damarsal içinde NO üretimini anlamaya başlamak için yeterli derinlik ve çözünürlükte görüntü mümkün kılar. İki foton mikroskop son zamanlarda doku, organ ve çünkü derinden düşük eşiğe ve yüksek sinyal duyarlılığı ile dokulara nüfuz üstün yeteneği bile tüm hayvan çalışmalarında uygulanmıştır. 1,2 illuminat iki foton uyarma dar spektrumluiyon odak noktası olan ve olmayan descanned dedektörlerin kullanımı iki foton mikroskopi geleneksel konfokal mikroskobu üstündür nedenleri vardır. Mikroskopisi nedeniyle oto-floresan ve konfokal iğne deliği içine out-of-focus ışık saçılması için gerekli doku derinlikte yüksek kaliteli görüntüler üretemez. Sonuç olarak, [Ca2 +] i sinyal ölçmek için iki foton mikroskopu kullanılarak bir yöntem ve yüksek bir çözünürlüğe ve düşük sinyal-gürültü oranına sahip olduğu gibi, tek tek kan taşıyıcı hücrelerin NO üretimi geliştirdik.

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	<tbody>
		<tr>
			<td>DAF-FM</td>
			<td>Life Technologies</td>
			<td>D-23842</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo 4 AM</td>
			<td>Life Technologies</td>
			<td>F14217</td>
			<td>500&#181;l in DMSO</td>
		</tr>
		<tr>
			<td>Pluronic F-68 solution</td>
			<td>Sigma-Aldrich</td>
			<td>P5556</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Name</td>
			<td>Tocris</td>
			<td>0665</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus upright microscope</td>
			<td>Olympus</td>
			<td>Fluoview FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>MaiTai HP DeepSee-OL&#160;</td>
			<td>Spectra Physics</td>
			<td></td>
			<td>Ti:sapphire laser 690nm &#8212; 1040nm</td>
		</tr>
		<tr>
			<td>Filters</td>
			<td>Olympus</td>
			<td>FV10-MRL/R&#160;</td>
			<td>495 to 540 nm&#160;</td>
		</tr>
		<tr>
			<td>25&#215; water-immersion objective lens</td>
			<td>Olympus</td>
			<td>XLPL25XWMP</td>
			<td>N.A. 1.05 and working distance 2 mm</td>
		</tr>
		<tr>
			<td>Slice Anchor grid&#160;</td>
			<td>Warner Instrument&#160;</td>
			<td>SHD-27LH</td>
			<td></td>
		</tr>
		<tr>
			<td>Other basic reagents&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

two-photon imaging of intracellular ca2+ handling and nitric oxide production in endothelial and smooth muscle cells of an isolated rat aorta

Kalsiyum vasküler dokularda çok sayıda fizyolojik süreçte önemli bir düzenleyicidir. En endotel ve düz kas fonksiyonları yüksek hücre içi kalsiyum ([Ca2 +] i) ve nitrik oksit (NO) değişikliklere bağlıdır. Amacıyla iki foton mikroskobu ile boyaları [Ca 2 +] i, NO ve alt molekülleri vazokonstriktörlere ve vazodilatörler yanıt olarak bir kan damarı tarafından işlenir, biz kalsiyum etiketleme geçerli yeni bir teknik (ya da NO-etiketleme) geliştirdi nasıl anlamak için İzole kan damarlarındaki kalsiyum taşıma (veya NO üretimini) ölçmek için. Burada anlatılan sıçan, etiket kalsiyum veya HAYIR endotel veya düz kas hücrelerinin içinde aort izole ve görüntü kalsiyum geçici (veya HAYIR üretim) fizyolojik aşağıdaki iki foton mikroskop kullanarak nasıl gösterir bir ayrıntılı adım-adım prosedürü ya da farmakolojik uyaranlar. yöntemi kullanmanın yararları çok yönlüdür: 1) aynı anda mümkündürly endotel hücreleri ve farklı uyarıcılara tepki olarak, düz kas hücreleri, hem de geçici kalsiyum ölçülmesi; 2) görüntü, endotelyal hücreler ve nativ ortamda düz kas hücrelerinin bir sağlar; 3) Bu yöntem hücre içi kalsiyum veya HAYIR değişiklikler ve hassas ölçümler için yüksek çözünürlüklü görüntüler üretir karşı çok hassastır; ve 4) tarif edilen yaklaşımı, bu reaktif oksijen türleri gibi diğer moleküller, ölçümü için uygulanabilir. Özetle, iki foton lazer emisyon mikroskobu uygulama geçici kalsiyum ve izole bir kan damarı endotel ve düz kas hücrelerinde NO üretimini yüksek kalitede nicel verilerin sağlanan ve vasküler fonksiyonları düzenleyen mekanizmaların anlayışımızı teşvik etmiştir izlemek için.

Doğru vasküler fizyoloji (damar genişlemesi ve vazokonstriksiyon) kalsiyum katkısını değerlendirmek için, bir protokol endotel hücreleri ve izole sağlam aortlarından düz kas hücrelerinin hem kalsiyum boyalar yüklemek için tasarlanmıştır. Şekil 1 &#39;de gösterilen kurulmuş, genel deney, izolasyon ve görüntüleme önce kabın hazırlanması için temel strateji gösterir. Kısaca, sıçan aort izolasyonundan sonra, yağ ve bağ dokusu temizlenmeli ve uzunlamasına ...

Watch this Scientific Journal Video about Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta at JoVE.com

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 ...

two-photon-imaging-intracellular-ca2-handling-nitric-oxide-production

Research

JoVE Journal

Biology

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

11.2K Görüntüleme.  Medical College of Wisconsin. 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. 

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

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

İki foton axotomy ve zaman atlamalı konfokal görüntüleme canlı zebrafish embriyolar

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Biyoloji

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Odak Ca 2 +</supDüz kas> Geçici Algılama

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

İnsan Umblikal Arter düz kas hücrelerinin izolasyonu (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

Farmakoloji Araştırma İzole Doku Banyo-uygulamalarda Düz Kas Fonksiyon Ölçümü

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Görüntüleme Yerel Ca<sup&gt; 2+</sup&gt; Kültürlenmiş memeli hücrelerinde sinyaller

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

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

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Bozulmamış Arterlerin endotel katmanı Nitrik Oksit ve Süperoksit En Yüz Algılama

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

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Kullanımı Kültür düz kas hücrelerinin sarkoplazmik retikulum içinde Kalsiyum Değişiminin Ölçülmesi FRET tabanlı Konfokal Görüntüleme

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Murin Koroner Damar düz kas hücrelerinin izolasyonu

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

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 ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Murin Aort Kırma Yaralanması: Verimli<emIn vivo</em&gt; Düz Kas Hücresi çoğalması ve Endotel Fonksiyon Modeli

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...