Name: culturing primary rat inner medullary collecting duct cells
Uploaded: 2013-06-21T14:00:00
Description: Watch this Scientific Journal Video about Culturing Primary Rat Inner Medullary Collecting Duct Cells at JoVE.com

支持这项工作是由德意志研究联合会（DFG; KL 1415/4-2 1415/3-2和KL）。

1。准备<ol><li>准备培养皿<ol><li>解冻胶原蛋白​​IV型O / N在4°C和0.1％无菌醋酸溶解。使用量取决于接种细胞的类型和数量的菜肴。使用2微克/厘米2。 </li><li>孵育至少1小时，于RT菜肴，并用蒸馏水（A. tridest）洗两次。 </li><li>允许菜肴正常干燥。 </li></ol></li><li>补充介质<ol><li>提高到500毫升培养基中添加1.75克葡萄糖，4.5 g / L的葡​​萄糖水平。为了调节介质600毫渗透摩尔，添加100mM NaCl的1...

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我们提出了一个详细的协议的准备和培养原代大鼠IMCD细胞。该方法产生高达21平方厘米的细胞从一个大鼠20。实验要求标准的细胞培养设备，并可以在大约6小时内由一个人进行。因此，这种做法是适合作为一个标准的实验室方法。 原代大鼠的IMCD细胞可以接种在培养皿不同大小，范围从96孔板到60毫米的菜肴。然而，环比增长384格式的程序需要优化。我们避免?...

A correction was made to <a href="http://www.jove.com/video/50366">Culturing Primary Rat Inner Medullary Collecting Duct Cells</a>. There was an error with an author's name. The author's last name had a typo and was corrected to: Enno Klussmann instead of: Enno Klussman

A correction to author list has been made for article Culturing Primary Rat Inner Medullary Collecting Duct Cells.

Erratum: Culturing Primary Rat Inner Medullary Collecting Duct Cells

在肾集合管主细胞，精氨酸加压素（AVP）控制水的重吸收，通过刺激插入水通道蛋白2（AQP2）到质膜。 AVP结合G蛋白偶联后叶加压素2型受体（V2R），刺激腺苷酸环化酶，从而cAMP的形成。启动该信号级联激活蛋白激酶A（PKA）。蛋白激酶A磷酸化水通道在丝氨酸256（S256），这是关键的触发再分配到质膜的细胞内小泡。膜插入沿渗透压梯度和微调体内水分的动态平衡，促进水的重吸收。 失调的AVP-介导的水的重吸收，由于像差的AVP分泌或AVP-激活的的信令原因或伴有严重的疾病。水的重吸收减少V2R或AQP2突变造成的，而导致肾性尿崩症海拔血液ATED血浆AVP水平与心血管疾病，如慢性心脏衰竭及抗利尿激素分泌综合征（SIADH）水的重吸收过多。 AVP-介导的水的重吸收的意义，不仅是因为它蕴涵在疾病。 AVP诱导易位AQP2息囊泡与细胞膜的融合代表了严格cAMP依赖的胞吐过程，这是目前不是很了解。为cAMP依赖的胞吐作用的其他例子是在肾脏和H +在胃中分泌的肾素分泌。因此，阐明AQP2易位肾主细胞的分子机制，不仅有助于理解相似的分子过程，在其他类型的细胞，但也可能与AVP-介导的水的重吸收障碍有关的疾病的治疗新疗法铺平道路。 êlucidating控制机制A​​QP2需要收集管主细胞模型。对于这样一些不同的哺乳动物肾细胞系都可用。然而，这些模型有几个缺点。在许多细胞系统中的水通道蛋白水平低（ 表1，M1，HEK293，COS-7，MDCK细胞，LLC-PK1）1-10，其他异位过度表达人类（MCD4，WT10）11,12或大鼠（CD8） 13 AQP2。在MCD4和COS-7细胞中的V2受体不表达。据我们所知，没有永久的细胞株作为一种模式，这是来自肾脏内髓集合管，肾AQP2表达最丰富的那部分，但是从皮质集合管1,11,13-16 。这种细胞模型，例如广泛使用的永生mpkCCD（ 例如 17），或最近建立的mTERT-CCD 14个细胞系。两种细胞系内源性表达AQP2和V2R但因为它们是来自最有可能含有皮质集合管内髓集合管（IMCD）细胞相比，不同的蛋白质组。例如，他们必须表达不同的Na +的运输系统。 在这里，我们提出了一个协议文化主髓集合管细胞表达V2R和AQP2内源性。这一模式，因此，代表着最密切的生理情况在肾集合管。建立文化，只需要标准的实验室设备其他实验室可以很容易地采用这种方式。

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company Catalog</td>
			<td>Number</td>
		</tr>
		<tr>
			<td>Collagen Type IV Mouse</td>
			<td>BD Biosciences</td>
			<td>356233</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>SIGMA</td>
			<td>H6254</td>
		</tr>
		<tr>
			<td>Collagenase type CLS-II</td>
			<td>Biochrom AG</td>
			<td>C2-22</td>
		</tr>
		<tr>
			<td>DMEM + GlutMAX</td>
			<td>Invitrogen GIBCO</td>
			<td>21885108</td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>SIGMA</td>
			<td>N4014</td>
		</tr>
		<tr>
			<td>Ultroser G</td>
			<td>Cytogen</td>
			<td>15950-017</td>
		</tr>
		<tr>
			<td>Non essential amino acids (NEA)</td>
			<td>Biochrom AG</td>
			<td>K0293</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Invitrogen GIBCO</td>
			<td>15710</td>
		</tr>
		<tr>
			<td>DBcAMP</td>
			<td>BIOLOG</td>
			<td>D009</td>
		</tr>
	</tbody>
</table>

culturing primary rat inner medullary collecting duct cells

精氨酸加压素（AVP）肾集合管主细胞，从而微调体内水分的动态平衡，促进水的重吸收。 AVP的后叶加压素V2受体（V2R）的表面上的细胞结合，从而诱导的cAMP合成。这刺激细胞信号传导过程中的水通道蛋白2（AQP2）的磷酸化的变化。蛋白激酶A phoshorylates AQP2，从而触发细胞内的囊泡AQP2易位到质膜促进水的重吸收原尿。从主细胞垂体AVP激活的信号畸变AVP释放可引起中央或肾性尿崩症，分别升高血浆AVP水平与心血管疾病，如慢性心脏衰竭及抗利尿激素分泌综合征。 在这里，我们提出了一个协议cultivati对原代大鼠内髓集合管（IMCD）细胞，快递V2R和AQP2内源性的。这些细胞是适用于阐明的分子机制，水通道的控制，从而发现新AVP-介导的水的重吸收的失调相关的疾病的治疗的药物靶点。髓集合管细胞是从大鼠肾脏内髓质和六到八天播种后用于实验。髓集合管细胞可以定期细胞培养皿，培养瓶和不同格式的微滴定板中培养，该过程只需要几个小时，适合标准的细胞培养实验室。

原代大鼠IMCD细胞培育成功，将导致在一个融合的单层播种后6-8天（ 图2）。每60毫米培养皿中有大约6×10 6细胞。这些细胞紧紧坚持培养皿，这些被涂上IV型胶原，基底膜成分18。因此，IMCD细胞将无法分离，即使在一些彻底的洗涤程序。高达80％的培养细胞表达出来的内源性和AQP2。这是主要的细胞，，而AQP2细胞缺乏被认为是闰细胞，非髓集合管细胞是来自于薄的四肢，?...

Watch this Scientific Journal Video about Culturing Primary Rat Inner Medullary Collecting Duct Cells at JoVE.com

Culturing Primary Rat Inner Medullary Collecting Duct Cells

精氨酸加压素（AVP）控制体内水分的动态平衡微调肾主细胞，通过促进水的重吸收。在这里，我们提出了一个协议的初级大鼠内髓集合管细胞AVP-介导的水的重吸收的分子机制的阐明适合种植。

原代培养大鼠内髓集合管细胞

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Research

JoVE Journal

Biology

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

在手术伤口的痛觉及炎症介质的收集和测量

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

科研

生物学

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

培养大鼠颈上神经节（SCG）的交感神经元的议定书

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

Primary Culture of Adult Rat Heart Myocytes

成年大鼠心脏心肌细胞的原代培养

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Primary Human Bronchial Epithelial Cells Grown from Explants

从外植体生长的首要人权支气管上皮细胞

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

小鼠气管基底细胞和粘膜下腺体的导管细胞分离

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Isolation and Primary Culture of Rat Hepatic Cells

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Method for Culturing Embryonic C. elegans Cells

A Method for Culturing Embryonic C. elegans Cells

一种用于培养胚胎<em&gt; C。线虫</em&gt;细胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...