We would like to thank Dr. Alengo Nyamay'antu and Dr. Ilse Timmerman for their advices about lentiviral packaging. This work is supported by an American Heart Association Scientist Development Grant 10SDG4170137.

<ol>
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The authors have nothing to disclose.

Neonatal sıçan izole edilmiş primer kardiyomiyositlerde uzun in vitro kardiyomiyosit fonksiyonlarını incelemek için kullanılmıştır. Bu protokol, ilk olarak 4 ° C&#39;de tripsin O / N ile sindirerek ve daha sonra saflaştırılmış kolajenaz, iki aşamalı bir enzim sindirimi yöntemi kullanılarak sıçan yavrularından neonatal kardiyomiyositlerde izolasyonu için bir yöntem tarif eder. Saflaştırılmış kolajenaz adımı kullanarak bir avantajı enzim aynı sınıf tüm izolasyon için kullanı...

İlköğretim sıçan yenidoğan kardiyomiyositler uzun vitro 1 kardiyomyosit fonksiyonlarını araştırmak için kullanılmıştır. Onlar birkaç iyi kurulmuş yöntemlerle 2-4 sıçan yavrular izole etmek kolaydır. En yaygın yöntem kolajenazı önce veya hücre izolasyonu için kalbin bağ dokusu sindirmek için tripsin kullanır. Araştırmacılar ayrıca yetişkin kemirgenler 5-8 yanı sıra yenidoğan farelerin 9,10 dan kardiyomiyositlerin izole etmek için yöntemler geliştirmişlerdir. Bu protokol, iki aşamalı bir prosedür kullanılarak enzim sindirimi, neonatal sıçan yavrularından kardiyomiyositlerde izole edilmesi için bir yöntem tarif eder. Tripsin ilk 4 ° C &#39;de O / N el ve 37 ° C sıcaklıkta ve sonra saf bir kolajenaz 4 ° C&#39;de tripsin O / N, kalp dokusu inkübasyonu sıcak bir enzim solüsyonu 2 ardışık inkübasyonlar kullanan yöntemleri ile karşılaştırıldığında hücre hasat için gerekli adımları azaltır. Buna ek olarak, saflaştırılmış co kullanarakllagenase daha basit bir enzimlere göre, çok partiye değişkenlik dolayısıyla daha tekrarlanabilirlik sağlayan, elimine edilebilir. , Belirli bir proteinin fonksiyonel çalışmalar, genellikle bir adenovirüs 11-13 ve / veya 14-16 lentivirüs kullanarak bir protein ifade sistemi kullanır. [Uyarıcı not] viral üretim ve manipülasyon NIH talimatlarına göre yapılmalıdır. Adenovirüs konakçı genomu içine entegre değildir. Bu bölünen hücreler ve bölünmeyen hücreleri de dahil olmak üzere pek çok hücre tipi, hem birincil hücre ve hücre serileri de transdüksiyon çok yüksek bir etkinliğe sahiptir. Bu, adenovirüs gen ifadesi için güvenilir bir vektör sağlar. Adenovirüs vektörü tarafından kodlanan proteinin yüksek seviyeleri transdüksiyonu takip eden 48 saat içinde gelişir ve bunlar birkaç hafta 17 sürebilir. Bununla birlikte, protein ekspresyonu için bir adenovirüs kullanımının bir dezavantajı olduğu bir yeniden birleştirici ade geliştirilmesiAdenovirüs karmaşık ve zaman alıcı hem de. Birçok araştırmacı rekombinant gen ifadesi için Lentivirüslerden dönüm edilmiştir neden bu dezavantajı kısmen açıklıyor. Adenoviral yapıları aksine, bir lentiviral yapı üreten hızlı ve kolaydır. Lentivirüsler genellikle her iki bölme ve bölünmeyen hücrelere adenovirüs daha düşük transdüksiyon verimliliği, sahip olmakla birlikte, bunlar bir konakçı genom içine entegre yok. Sonuç olarak, kalıt aktarılan genin ifadesi için adenovirüsler daha lentivirüsler daha kararlıdır. Nedeniyle mikroskopi alanındaki teknolojik gelişmeler, bu rekombinant proteinleri ifade eden hücrelerin, canlı hücre görüntülerini yakalamak için çok daha kolaydır. Bu bile edinimi video oranı hızlarda geçerlidir. Bu durum araştırmacı ilgilenilen proteinin, özellikle işlevsel değişiklikler, gerçek zamanlı olarak etki hücreyi belirlemek için izin verir. Konfokal dönen disk mikroskop o liv için optimal bir tekniktir yapmak birçok önemli özelliklere sahiptirE hücre görüntüleme 18,19. Aynı anda nokta tarama konfokal mikroskoplar çok daha az lazer güç kullanırken Yokogawa eğirme disk, çok daha hızlı görüntü elde etmek için izin verir. Bu özelliklerin her ikisi de lazer örnekler aynı zamanda geçtiği çok sayıda eş odaklı delik içeren dönen bir disk, kaynaklanmaktadır. Satın alma sırasında disk kendisini hızla ve sürekli olarak 18-20 spin. Mikroskop otofokus sistemi kullanarak, istikrarlı odak uzun süreler boyunca devam edilir. Bu araştırmacılar, iyi odaklanmış canlı hücre görüntü almasına izin veriyor. Edinsel Görüntülerin bir film dosyası olarak oynatılır. Görüntüleri, ImageJ 21,22, FIJI 23 ya da başka ticari olarak temin edilebilen yazılımı gibi görüntü analizi yazılımı kullanılarak analiz edilmiştir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1 scissors for decapitation</td>
			<td>WPI</td>
			<td>501749</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>1 fine scissors for heart isolation and chopping</td>
			<td>WPI</td>
			<td>14393</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>2 fine forceps (Dumont No. 5)</td>
			<td>Sigma</td>
			<td>F6521</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>Three sterilized 10 cm plastic dishes</td>
			<td>Sigma</td>
			<td>CLS430165</td>
			<td>for hearts isolation</td>
		</tr>
		<tr>
			<td>3.5 cm glass bottom culture dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-20-C</td>
			<td>for final plating of cardiomyocytes for future live cell imaging. micro-Dishes from ibidi are an acceptable alternative.</td>
		</tr>
		<tr>
			<td>3.5 cm glass bottom culture dishes</td>
			<td>MatTek</td>
			<td>P35G-0.17-14-C</td>
			<td>for TIRF or high resolution image</td>
		</tr>
		<tr>
			<td>Ethanol solution, 70 % (v/v) in water</td>
			<td>Sigma</td>
			<td>E7148</td>
			<td></td>
		</tr>
		<tr>
			<td>2% gelatin</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>One sterilized 10 cm plastic dish</td>
			<td>Sigma</td>
			<td>CLS430165</td>
			<td>for trypsinization</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>parafilm</td>
			<td>Sigma</td>
			<td>P7543</td>
			<td></td>
		</tr>
		<tr>
			<td>Two 10 cm plastic cell culture dish</td>
			<td>Sigma</td>
			<td>CLS430165</td>
			<td>for selection</td>
		</tr>
		<tr>
			<td>Auto pipette</td>
			<td>Drummond Scientific&#160;</td>
			<td>4-000-300</td>
			<td>for trituration</td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Cellometer, automated cell counter</td>
			<td>nexcelom</td>
			<td></td>
			<td>to check and count cells</td>
		</tr>
		<tr>
			<td>&#160; Microscope and Hematocytometer</td>
			<td>any brand</td>
			<td></td>
			<td>to check and count cells</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>invitrogen</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Sanyo</td>
			<td>MCO-19AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>incubating orbital shaker</td>
			<td>Sigma</td>
			<td>Z673129</td>
			<td>to shake heart tissue with collagenase at 37 C at 170-200 rpm</td>
		</tr>
		<tr>
			<td>10 mg/ml BrdU solution</td>
			<td>BD Pharmingen</td>
			<td>550891</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose</td>
			<td>invitrogen</td>
			<td>41965039</td>
			<td>Mix medium as indicated in the protocol and warm before use</td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>invitrogen</td>
			<td>31095029</td>
			<td>Mix medium as indicated in the protocol and warm before use</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>invitrogen</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)&#160;</td>
			<td>invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Section 2 lentiviral transduction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>three packaging plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;pRSV-Rev</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>The lentiviral transfer vector, pLVX-IRES-Puro</td>
			<td>Clontech</td>
			<td>632183</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM (serum-free medium)</td>
			<td>invitrogen</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>transfection reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; polyethyleneimine&#8220;Max&#8221;, (Mw 40,000) - High Potency Linear PEI (Equivalent to Mw 25,000 in Free Base Form)&#160;</td>
			<td>Polysciences</td>
			<td>24765-2</td>
			<td>It can be substituted with X-tremeGENE 9 from Roche</td>
		</tr>
		<tr>
			<td>&#160; X-tremeGENE 9</td>
			<td>Roche</td>
			<td>6365779001</td>
			<td>substitute for PEI as transfection reagent</td>
		</tr>
		<tr>
			<td>chloroquine</td>
			<td>Sigma</td>
			<td>C6628</td>
			<td>dissolve in water and make 100 mM stock solution. Inhibition of endosomal acidification can be achieved with 10-100 &#956;M Chloroquine.</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml Luer-Lok syringe, sterilized</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 um filters</td>
			<td>Sigma</td>
			<td>F8677</td>
			<td>use only cellulose acetate or polyethersulfone (PES) (low protein binding) filters. Do not use nitrocellulose filters. Nitrocellulose binds surface proteins on the lentiviral envelope and destroys the virus.</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>dissolve in water and make 8mg/ml stock solution, then filter it to sterilize.</td>
		</tr>
		<tr>
			<td>polyethylene glicol 6000</td>
			<td>Sigma</td>
			<td>81260</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Section 3 Adenoviral transduction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>Some 10 cm cell culture dishes</td>
			<td>Sigma</td>
			<td>CLS430165</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Microplate with lid, flat-bottom, tissue culture, sterile</td>
			<td>BD Falcon</td>
			<td>353072</td>
			<td>for titration</td>
		</tr>
		<tr>
			<td>Multichannel piptte, 10-100 ul, 8-channel</td>
			<td>eppendorf</td>
			<td>3122 000.035</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Section 4 live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disk confocal microspopy</td>
			<td>PerkinElmer</td>
			<td>L7267000</td>
			<td></td>
		</tr>
		<tr>
			<td>a temperature controlled chamber</td>
			<td>any brand</td>
			<td></td>
			<td>to keep temperature at 37 C</td>
		</tr>
		<tr>
			<td>a CO2 environmental system</td>
			<td>any brand</td>
			<td></td>
			<td>optional to maintain CO2 concentration optimal</td>
		</tr>
		<tr>
			<td>Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; CO2 Independent Medium, No Glutamine</td>
			<td>invitrogen</td>
			<td>18045-054&#160;&#160;</td>
			<td>for long time time-lapse imaging</td>
		</tr>
		<tr>
			<td>&#160; DMEM, High Glucose, HEPES, no Phenol Red&#160;</td>
			<td>invitrogen</td>
			<td>21063-029</td>
			<td>for long time time-lapse imaging</td>
		</tr>
	</tbody>
</table>

live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy

Kolayca tek bir işlem içinde çok sayıda izole edilebilir, çünkü Birincil sıçan neonatal kardiyomiyositlerde vitro Kardiyovasküler araştırmalarda temel yararlıdır. Nedeniyle mikroskop teknolojisindeki ilerlemeler bu hücrelere fototoksisite ilişkin en az endişe ile gerçek zamanlı olarak hücresel olayları araştırmak amacıyla canlı hücre görüntülerini yakalamak için nispeten kolaydır. Bu protokol hücrenin özelliklerini modüle Lentiviral ve adenoviral transdüksiyon izleyen bir konfokal dönen disk mikroskop kullanılarak primer sıçan yenidoğan kardiyomiyositlerin canlı hücre timelapse görüntüleri çekmek için nasıl açıklar. Virüs iki farklı uygulama daha kolay iki farklı genler için uygun bir iletim hızı ve ekspresyon seviyelerini elde etmek için yapar. Peki odaklı canlı hücre görüntüleri uzun süreler boyunca istikrarlı odak tutar mikroskop otofokus sistemi kullanılarak elde edilebilir. Bu yöntemde, dışsal mühendisinin işlevlerikültürlenmiş primer hücrelerde ifade ed proteinlerin analiz edilebilir. Buna ek olarak, bu sistem siRNA&#39;lar kullanımı yoluyla hem de kimyasal modülatörlerinin genlerin fonksiyonlarını incelemek için kullanılabilir.

Teknik, lentivirüs encoding EGFP (gelişmiş yeşil floresan protein)-etiketli Cx43 (Connexin43) veya hücrelerde EGFP-etiketli proteinleri ifade etmek için kullanılan bir mutasyona uğramış Cx43 32 ve bir adenovirüs kodlama FGFR1DN (fibroblast büyüme faktörü reseptörü 1 baskın negatif açıklamak için ) hücrede 33-35 FGF sinyalizasyon kapatmaya kullanıldı. Kardiyomiyositlerde izolasyonu, aşağıdaki üç gün, izole edilmiş kardiyomiyositlerde hücrelerinde EGFP-etiketli protein...

Watch this Scientific Journal Video about Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy at JoVE.com

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

İlköğretim Rat Yenidoğan kardiyomiyositlerin Canlı Hücre Görüntüleme Adenoviral izlenerek ve Lentiviral İletimi Confocal Disk Mikroskopi Spinning kullanma

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

live-cell-imaging-primary-rat-neonatal-cardiomyocytes-following

Research

JoVE Journal

Biology

13.4K Görüntüleme.  Max-Planck-Institute for Molecular Biomedicine and Institute of Cell Biology. This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Video: Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy