Name: single-cell microinjection for cell communication analysis
Uploaded: 2017-02-26T13:00:00
Description: Watch this Scientific Journal Video about Single-cell Microinjection for Cell Communication Analysis at JoVE.com

The authors dedicate this paper in honor of Prof. Gilberto Oliveira-Castro who introduced research in intercellular communication by gap junctions in Brazil. This work was funded by Capes, CNPQ and Faperj.

<html> Hücrelerin hazırlanması 1. <ol><li> Bir timüs epitelyal hücre hattı (IT76M1) ya da hücrenin bir kültür muhafaza bir kuluçka makinesi içinde, test edilecek (37 ° C /% 5 CO2). </li><li> PBS 1x hücreleri yıkayın (bu öğeyi 3x tekrarlayın). </li><li> 5 dakika için bir hücre için tripsin ekleyin. </li><li> tripsin ve santrifüj (5 dakika boyunca 800 x g) ile hücrelerin% 10 FBS (fetal inek serumu) ile (Madde 1.3 ilave tripsin hacminin iki kez) orta ekleyin. </li><li> Bir hemasitometre h...

<ol>
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Hücrelerarası kanallar tarafından geçirgen 16 gerekmesine rağmen, fonksiyonel hücrelerarası boşluk kavşak varlığını, membran geçirmeyen izleyiciler, kullanımını doğrulamak için. Fluoresein, ilk fluoresan boya hücre-hücre bağlama 22, sigara kavşak zarlar 3 ila geçirgendir ve bu nedenle, Lucifer sarı boya 15 ile ikame edilmiş gözlemlemek. Şu anda, florasan izleyiciler birçok farklı türleri arasın...

Gap junction komşu hücrelerden 1 arasında bağ kurar izin hücrelerarası kanallardır. Bu iletişim, her biri içi kanal oluşturmak üzere bir connexon ya hemichannel katkıda bulunur, iki ya da daha fazla komşu hücreler, bağlanır. Memeli hücrelerinde, connexon sitoplazma 2 içinde dört transmembran ve bir C ve N terminal ile altı connexins monomer oluşturulmuştur. Ara bağlantılar, yalnızca iyonlar, ikinci haberciler ve küçük metabolitlerin akışına izin vermek, ama aynı zamanda sinaptik iletim kalp kasılması, hücre büyümesi ve farklılaşması, 3, 4, 5, 6 gibi birçok fizyolojik işlemlerde, hücresel haberleşme birçok şekilde katkıda 7, 8. Buna ek olarak ara bağlantılar ile ilişkilendirilmiştirkanser 9, 10, müsküler atrofi 11, bazı genetik hastalıklar ve hastalıkların 12 demiyelinizan dahil olmak üzere birçok hastalık. Arası karışma Bu tür çeşitli yöntemlerle 13, 14, 15, 16 ile değerlendirilebilir. Bu yazıda canlı hücrelerin boşluk bağlantıları aracılığıyla hücresel iletişim görselleştirmek için Lucifer Sarı bir tek hücreli mikroenjeksiyon nasıl gerçekleştirileceğini gösterir. Bu hücreler ve mikropipet, mikromanipülatör kullanımını ve timik epitelial hücre çizgisinde Lucifer sarı boyadan enjeksiyonun hazırlanması için nasıl ele. Genellikle, bu deney prosedürü, boyası yüklendi hücreye bağlanan hücrelerin ortalama analiz edilebilir. Buna ek olarak, bu yöntem, boşluk altında molekül ağırlığına sahip diğer floresan boyalar ile birlikte kullanılabiliryaklaşık 1000 dalton olan kavşak kesme.

<table border="0" cellpadding="0" cellspacing="0" width="1228">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Lucifer yellow</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">L0259</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Lithium Chloride</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">L4408</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PBS tablets</td>
			<td style="width:71px;">Sigma&#160;</td>
			<td style="width:137px;">P4417</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">RPMI</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">R4130</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Bovine fetal serum</td>
			<td style="width:71px;">Cultilab</td>
			<td style="width:137px;"></td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Trypsin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">T4799</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:137px;">TE-2000</td>
			<td>For microinjection experiments, one needs an inverted fluorescence microscope and &#64257;lters for &#64258;uorescent microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vibration-insulated table&#160;</td>
			<td style="width:71px;">Newport</td>
			<td style="width:137px;">VH3036W-OPT</td>
			<td>A vibration-insulated table is needed to protect the experiments from vibration and avoid cell damage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td style="width:71px;">Narishige</td>
			<td style="width:137px;">MMO-203</td>
			<td>This equipment allows precision adjustments of the micropipette, which is needed for cell micro injection.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;">Current Generator&#160;</td>
			<td style="width:71px;">Digitimer</td>
			<td style="width:137px;">DS2</td>
			<td>To produce the dye flow through the micropipette, a current below one nano ampere was given using a current generator with an electrode inside the micropipette or an amplifier which has a capacitance compensation circuit (old electrometer) or current injection functions of new patch clamp amplifiers, and the ground wire submersed in the plate dish. Alternatively, the dye can be injected by a pneumatic microinjector, following the factory recommendations.&#160;&#160;&#160;</td>
		</tr>
	</tbody>
</table>

single-cell microinjection for cell communication analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

Bu hücreler 43 21 konneksin oluşan fonksiyonel gap junction ifade etmek açıklandığı gibi timik epitel hücre çizgisi IT-76MI gap junction ile boya kaplin değerlendirmek için kullanıldı. pipet ucu altındaki bir hücreye uygulandığında Şekil 1, Lucifer Yellow enjeksiyonunu göstermektedir. Birkaç dakika sonra, bağlanmış hücreler boşluk bağlantıları ile floresan boya difüzyonu gösteren floresan (yıldız) haline gelir. hücre ve zaman şek...

Watch this Scientific Journal Video about Single-cell Microinjection for Cell Communication Analysis at JoVE.com

Single-cell Microinjection for Cell Communication Analysis (Video) | JoVE

Biz canlı hücrelerin boşluk bağlantıları aracılığıyla hücresel iletişim görselleştirmek için Lucifer Sarı bir tek hücreli mikroenjeksiyon gerçekleştirin ve bazı yararlı ipuçları sağlamak için nasıl burada açıklamak. Biz bu kağıt nedeniyle fonksiyonel gap junction hücresel bağlantı derecesini değerlendirmek için herkese yardımcı olacağını bekliyoruz. Burada tarif edilen her şey 1,000 Dalton altında molekül ağırlığına sahip diğer floresan boyalar için uyarlanmış, ilke olarak, olabilir.

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