Finansman &quot;Progetto bir Concorso Fondazione IRCCS Ospedale Maggiore Policlinico 2009-2010&quot; MD, ve Associazione Bambino Nefropatico ABN Onlus, Milano tarafından sağlandı

Tüm hayvan deneyleri Ulusal ve bilimsel amaçlar için kullanılan ve inceledim ve Milano Üniversitesi etik komitesi tarafından onaylanmış hayvanların korunmasına ilişkin Avrupa yürürlükteki mevzuata göre yapılmıştır. İlköğretim Osteoblastlar 1. İzolasyon Bir yöntem olup, küçük bir modifikasyonla, Dodig ve arkadaşları 14 tarafından tarif edilen prosedür takip eder. <ol><li> % 0.1 kollajenaz P ve% 0.05 ekleyerek, orta sindirilmesi hazırlayın HBSS ortamı (Hanks Dengelenmiş Tuz Çözeltisi, herhangi bir kalsiyum, magnezyum, ya da fenol kırmızı) için (% 2.5 tripsin 10X, fenol kırmızı başlayarak) tripsin. </li><li> 3-4 gün eski fareler kullanın (prosedür 1 fare gibi kaç uygulanabilir). </li><li> Başları kesilerek kurban. % 70 etanol ile baş Sprey ve buz üzerinde tutmak. Cımbız ve diseksiyon / cerrahi makas ile deri ve kas tabakaları çıkarın. </li><li> Yavaşça parietal kemikleri kaldırmak veiki devrede de gol sagital sütür izleyerek onları ayrı. Emin kemikleri sütür ve herhangi yapışık doku ücretsiz yapmak ve prosedür tamamlanana kadar HBSS ortamı içeren plakalarda bireysel kemik parçaları yerleştirin. </li><li> HBSS atın ve her bir ekle 37 ° C de 15 dakika boyunca ortam sindirerek </li><li> Süpernatant atın. </li><li> Tekrar 37 ° C&#39;de 15 dakika boyunca orta sindirerek ekleme </li><li> Bu kez süpernatan kaydetmek ve% 10 FBS (Fetal Sığır Serumu) ve% 1 streptomisin / penisilin ile desteklenmiş a-MEM (Minimum Essential Medium) yerleştirin. </li><li> Tekrar 1.7 ve 1.8 2x adımları. </li><li> 5 dakika boyunca 425 g&#39;de santrifüj ile üç toplanan fraksiyonları, spin hücreleri havuzda toplayın, süpernatantı atmak hücre pelletini için katkılı a-MEM içinde 3 ml ekleyin ve transferi 25 cm2 şişeye. </li><li> Enkaz ve ölü hücreleri çıkarmak için, 24 saat sonra ortam değiştirin. </li><li> 50 ug / ml, askorbik asit (AA) ve gly 3 mM eklemegliserin 2-fosfat disodyum tuzu, alfa-MEM ortamına hidrat (GP). </li><li> Bundan sonra, her gün (alfa-MEM artı AA / GP) orta değiştirmek. </li></ol> 2.. ATRA Protokol <ol><li> Etkisiz hale getirilmesini önlemek için, karanlık bir odada ATRA (all-trans retinoik asit) tüm deneyler. </li><li> HBSS ortamı içinde, 1X Tripsin çözeltisi hazırlandı ve kullanılana kadar 37 ° C&#39;de tutun. </li><li> Birlikte akan hücreler arasındaki orta çıkarın ve dikkatli bir şekilde HBSS ile yıkayınız. </li><li> 1 ml tripsin ekleyin, yavaşça tüm hücreler, tripsin ile kaplı izin vermek için şişeyi döndürmek ve 37 ° C&#39;de 3 dakika boyunca inkübe </li><li> Bir ters mikroskop altında hücre gerçek ayrılması için kontrol edin. </li><li> Tripsin inaktive alfa-MEM ortam maddesi 3 ml ilave edilir. </li><li> Hücreleri yeniden elde etmek ve 425 g&#39;de 15 dakika boyunca santrifüj. </li><li> Taze ortam içinde hücre pelletini. </li><li> Bir hemositometre sayım odasında bir damla yerleştirin, birkaç dakika boyunca yerleşmek için izin ve ce saymakll numarası. </li><li> 10,000 hücre / alfa-MEM plus AA / GP ile 2 cm &#39;lik bir yoğunlukta 25 cm2 şişelerde yer hücreler. Hücre yoğunluğu hücre süreçlerinin ve hücreler arası temasların optimum gelişimi ile ilgili olduğunu unutmayın. Bir başlangıç ​​yoğunluğu daha yüksek 10,000 hücre / cm 2 hücre süreçlerini uygun gelişimine zarar, ancak daha düşük hücre sayıları nedeniyle arası temasların olmadığı için, prematüre hücre ölümüne neden olabilir. </li><li> Her 24 saat ortama 5 uM ATRA ekleyin. </li><li> Morfolojik değişiklikler genellikle 48 saat sonra gözlenir: morfolojisini değerlendirmek için günlük hücreleri izleyin. Ilk 24-48 saat sonra, hücreler hala çoğaldığında,, yukarıdaki yoğunlukta replate. Olgun osteositler homojen bir popülasyon 4-5 gün sonra da rastlanmaktadır. Ihtiyaçlarına bağlı olarak, 12-15 gün öncesine kadar, herhangi bir zaman noktasında hücreleri kullanır. </li><li> Hücreleri (örneğin lamelleri ya da plakalar gibi) başka destekler üzerine plakalanır edildiği zaman, yukarıda yoğunluğu muhafaza emin olmak vekullanıma kadar ATRA her 24 saatte eklemeye devam. Deneyden önce en az 24 saat boyunca yeni destek uymak hücreleri bırakın. </li></ol> 3. İmmünoboyama <ol><li> Immün için, lamelleri hücreleri plaka ve (Burry 15 örneğin bakınız) kuruldu metodolojileri izleyin. </li><li> Örneğin dolaylı immünofloresans için, 30 dakika boyunca, oda sıcaklığında, karanlıkta, ikincil floresan etiketli antikor ile daha sonra, nemli bir bölmede 1 saat boyunca oda sıcaklığında primer antikor ile inkübe sekans,% 4 paraformaldehit veya soğuk aseton içinde çözmek nemlendirilmiş bir odada. </li><li> Nükleer counterstaining için DAPI veya analogları kullanın ve orta anti-solmaya montaj. </li></ol> 4. Alizarin Red Boyama ve Ekstraksiyon <ol><li> Lamelleri hücreleri plaka. </li><li> 10 dakika boyunca% 70 etanol içinde lamel içine daldırarak hücreleri sabitleyin. </li><li> Alizarin kırmızı boyama çözeltisi bir damla (1 mg / ml, pH 5.5) ile hücrelerin Kapak30 dakika karıştırıldı. </li><li> Musluk suyu ile lamelleri yıkayın. Cam slaytlar gliserol bir damla ile montaj ve dik bir 10X olarak mikroskop ve 20X büyütme ile görüntü alabilir. </li><li> Yukarıdaki prosedür sonra, musluk suyu içindeki slaytlar daldırarak lamelleri geri. </li><li> Bir oyuklu bir plaka içerisinde her bir lamel koyun. </li><li> % 60 perklorik asit (miktar tamamen hücreleri karşılamak için yeterli olması gerekir) ekleyin ve hafif çalkalama ile 4 ° C&#39;de O / N bırakın. </li><li> 450 nm dalga boyunda spektrofotometri ile süpernatantın optik yoğunluk okuyun. </li><li> Hücre sayısı değerlerini normalize etmek için, 10 dakika boyunca bir hücre preparatının kapsayacak şekilde Janus Green (PBS içinde% 0.2 çözelti) ekleyin. </li><li> Ultra-saf su ile dikkatlice yıkanır. </li><li> 0.5 N HCI (miktar tüm hücreleri karşılamak için yeterli olması gerekir) ekleyin ve bir kaç saniye boyunca elle hafifçe çalkalanır. </li><li> Spektrofotometre ile 615 nm&#39;de emicilik ile oku. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Son yıllarda osteosit kemiğin en merkezi hücre olarak ortaya çıkmıştır. Araştırma gelişmeler kademeli kemik hastalıklarının çeşitli için yeni ve daha iyi tedavi tasarımı için çok büyük bir değer olan, daha önce ya da umulmadık kanıtlanmamış osteosit özellikleri, bir çok açıklayıcıdır. Bununla birlikte, osteosit biyoloji araştırılması osteosit benzeri hücre hattı MLO-Y4 yayınlanmadan önce osteoblastlar, uzun bir süre boyunca taşıyıcı hücre olarak kullanılmış olduğu bi...

Kemik hücresinin, en bol kemik hücre tipi, derin iskeleti içinde yer alan büyük ölçüde dallanmış hücreler, terminal olarak farklılaşmış bulunmaktadır. Olgun osteositler ait hücre vücut kemik boşluklar içerdiği ve çeşitli şekillere sahip olan; yuvarlak osteositlerde trabeküler kemikte 1 daha sık iken uzun hücre organları ile osteositlerde, kortikal kemik bulunur. Dallı dendritler diğer kemik hücresinin ile değil, aynı zamanda diğer kemik hücre türleri, kemik iliği, kan damarları ve ilgili perisitlerin ile sadece birden fazla kişi karmaşık hale getiren bir ağ-yapının oluşturulması, kanalcığı adı verilen küçük kanallarda hücre gövdesinden uzanan ve bulunur. Boşluklar ve kanalcık içerdiği interstisyel sıvının sayesinde, osteositler nedenle lokal hem de sistemik etkinlik, ya da tam tersi davranışları hem lokal hem de sistemik değişikliklerle 2 ile kontrol edilebilir, sadece etkileyebilir, aynı zamanda sonuçta dolaşım sistemine bağlanmıştır. kemik hücresinin çalışması son zamanlarda, bu tür doku-ve hücreye özel transgenik hayvanların üretimi, güçlü mikroskopi teknikleri ve yüksek verimli tarama moleküler 3,4 kullanımı gibi çeşitli teknik ilerlemeler, sayesinde ivme kazanmıştır. Bununla birlikte, bu hücrelerin bilgisi esas olarak vitro modellerde yeterli göreli azlığı, yine eksik. Aslında osteositlerde nedeniyle derin konumu elde etmek ve kültür korumak için her zaman zor olmuştur, ve böyle bir farklılaşmış hücre tipini karakterize çoğalma düşük seviye var. Genellikle düşük verimli olduğu hücre ve hatta birkaç kirletici fibroblast hızla osteositler 8 büyümek doyurmaya riski taşıyan da yıllar boyunca, bir dizi yöntem, birincil kemik hücresinin 5-7 izole etmek için geliştirilmiştir. Bu nedenle, in vitro çalışmalarında en deneysel köklü osteosit hücre l hakkında şu ana kadar yapılmıştırine MLO-Y4 9. Ek in vitro metodolojiler bu hücreleri okuyan olasılığını artırabilir ve osteosit biyoloji ve patofizyolojinin analizini artırabilirsiniz. Büyük ölçüde kabul edilmesi, bu tür yöntemler çoğaltmak kolay olmalı ve hücre olgunlaşmasını ulaşmak için özel aletler ya da çok uzun bir süre gerektirecektir olmaz. Birincil hücreleri varsa da önemlisi, bu mümkün transgenik hayvanların yararlanmak olur. Yakın zamanda osteoblast hücre hattı MC3T3-E1 ve retinoik asit ile tedavi, birincil osteoblastların osteositler 10 morfolojik ve moleküler özellikler taşıyan dallanmış homojen bir hücreler nüfus gelişimine yol açan hızlı bir fenotip değişikliklerini oluşturmaktadır anlatılmıştır. All-trans retinoik asit (ATRA), nükleer retinoik asit reseptörleri (RAR&#39;lar) bağlanmak suretiyle gen transkripsiyonunu düzenler A vitamini aktif bir metabolizma ürünüdür. RAR&#39;larsonuçta retinoik asit (RA)-duyarlı hedef genler 11 modülasyonu yol açan, retinoid X reseptörleri (RXR&#39;ler) ile heterodimerler olarak DNA&#39;ya bağlanır. ATRA gibi nöronal hücre 12 ve 13 gibi diğer podocytes dallanmış hücreler, aralarında, çeşitli hücre tiplerinin farklılaşmasını ve olgunlaşmasını modüle ettiği gösterilmiştir. Burada tarif edilen yöntem, birincil osteoblastların için ATRA eklenmesi dayanmaktadır. Etkili olmak için, ATRA tanımlanmış bir yoğunlukta kaplama hücreleri üzerinde kesin bir olgunlaşma safhasında ilave edilmesi gerekir; deneyimlerimiz bu koşullar osteoblast gelen osteositlere için fenotip anahtarını elde etmek için önemlidir.

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			<td height="20" style="height:20px;width:283px;">Name</td>
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			<td style="width:183px;">comments</td>
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			<td height="20" style="height:20px;">Collagenase P</td>
			<td>Roche Applied Science</td>
			<td>11213857001</td>
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			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco, Life Technologies</td>
			<td>15400054</td>
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			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco, Life Technologies</td>
			<td>14175129</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alpha-MEM</td>
			<td>Invitrogen, Life Technologies</td>
			<td>22571038</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin/Penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol 2-phosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATRA</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>Protect ATRA from light</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Dissolve in PBS and filter before use. Work always under a chemical hood.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>32670</td>
			<td>Can be added to the secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Janus Green</td>
			<td>Sigma-Aldrich</td>
			<td>201677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Perchloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>176745</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fluorsave</td>
			<td>Calbiochem - Merck</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #7, 0.40 x 0.5mm tips</td>
			<td>World Precision Instruments</td>
			<td>501981</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #4, 0.40 x 0.45mm tips</td>
			<td>World Precision Instruments</td>
			<td>501978</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Scissors, straight,10cm curved</td>
			<td>World Precision Instruments</td>
			<td>14394</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Scissors, 14 cm, straight, S/S</td>
			<td>World Precision Instruments</td>
			<td>501218</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Culture flasks</td>
			<td>Corning</td>
			<td>430168</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Well-plates</td>
			<td>Corning</td>
			<td>3335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermanox coverslips</td>
			<td>Thermo Scientific Nunc</td>
			<td>12-565-27</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microscope</td>
			<td>Zeiss Apotome</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">spectrometer</td>
			<td>Safas Xenius</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts

<html> Osteosit kültürleri için olan ihtiyaç iyi kemik araştırmacıların eden bilinmektedir; Birincil osteositlere izolasyonu zordur ve düşük hücre sayıları üretmektedir. Bu nedenle, en yaygın olarak kullanılan hücre sistemi osteosit benzeri MLO-Y4 bir hücre hattıdır. Burada tarif edilen yöntem, morfolojik ve moleküler osteosit özelliklere sahip dallanmış homojen bir hücreler popülasyonunu üretmek üzere retinoik asidin kullanılması anlamına gelir. Fare kafatası alınan osteoblastların izole edildikten sonra, bütün-trans retinoik asit (ATRA) hücre ortamına ilave edilir ve hücre izleme ters döndürülmüş bir mikroskop altında her gün yapılır. Üretim morfolojik değişimler tedavi ve farklılaşma 2 gün sonra tespit edilebilir dendrit hücre dışı matrisi üretme yeteneği kaybı, osteoblast belirteçlerin aşağı-regülasyonu ve ilerleyici gelişmesiyle birlikte, 5 gün içinde tamamlanır yukarı-regülasyonu osteosit özgü moleküllerin. içeriği &quot;&gt; günlük hücre izlenmesi için birincil hücrelerin doğal değişkenlik gereklidir ve protokol, farklı fare türlerinden elde edilen ve transjenik modelleri uygulanan hücrelere, minimal değişiklik gösteren uyarlanabilir. Yöntem, gerçekleştirilmesi kolaydır ve özel aletler gerektirmez, bu yüksek oranda tekrarlanabilir olduğunu ve hızlı bir şekilde sınırsız biyolojik uygulamalarda bu hücrelerin kullanımına izin veren, hücre dışı matrisin tam yokluğunda olgun osteosit nüfus oluşturur.

Sonuçlar 5 ila 10 bağımsız deneyler için kullanılmıştır. Hücre Morfoloji AA / GP birincil hücreler çoğunlukla arnavut kaldırımı gibi özellikler, olgun osteoblastlar özelliği var tedavi. Serpiştirilmiş dallanmış hücreleri (Şekil 1A kırmızı oklarla gösterildiği gibi) büyük olasılıkla bir kemik hücresinin temsil ettiği, bulunabilir. ATRA tedavisi ile, hücreler hızla genellikle 2 gün sonra...

Watch this Scientific Journal Video about Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts at JoVE.com

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Retinoik asit ile osteoblastların primer fare tedavisi kemik hücresinin morfolojik ve moleküler özellikler taşıyan dallanmış homojen bir hücreler popülasyonu üretir. Yöntem, elde edilmesi ve kültürü birincil kemik hücresinin korumanın zorluğu üstesinden gelir ve transjenik modelleri türetilen hücreler çalışma avantajlı olabilir.

İlköğretim Fare Osteoblastlar gelen osteositlere Kültürler Edinme Retinoik Asit Uygulama

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

application-retinoic-acid-to-obtain-osteocytes-cultures-from-primary

Research

JoVE Journal

Biology

13.3K Görüntüleme.  Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.  Retinoik asit ile osteoblastların primer fare tedavisi kemik hücresinin morfolojik ve moleküler özellikler taşıyan dallanmış homojen bir hücreler popülasyonu üretir. Yöntem, elde edilmesi ve kültürü birincil kemik hücresinin korumanın zorluğu üstesinden gelir ve transjenik modelleri türetilen hücreler çalışma avantajlı olabilir. 

Video: Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Geç Stage Drosophila pupa Brain İlköğretim Nöronal Kültürler

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Biyoloji

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Kemirgen Yenidoğanlar İlköğretim grubuyla orta beyin dopamin Hücre Kültürleri

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Kemirgenler itibaren İlköğretim Yetişkin Fibroblast Kültürleri kurulması

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Birincil Hücre Kültürleri Drosophila Gastrula Embriyolar

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

Fare Kalpten İlköğretim Melanocyte gibi Hücreleri Ayırma

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

İlköğretim İnsan fibroblast Kültürler gelen naif Erişkin Kök Hücre İzolasyon

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

Fare ve İnsan Kolon Doku Primer miyofibroblastlar İzolasyonu

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Bazal Omurgalı soylarından İlköğretim andMyogenic Öncü Hücre / Miyoblast Kültürlerinin Hazırlanması

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.