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100mM的尿素，分别由200毫渗透摩尔，100毫渗透摩尔，增加渗透压。 </li><li>到600 mosmol DMEM培养基中添加1％非必需氨基酸，1％ultroser，500μM的二丁酰环腺苷酸（dbcAMP），20 U / ml的制霉菌素和0.25微克/毫升庆大霉素（1:200稀释的原液的浓度为50微克/毫升）新鲜的一天的准备。 </li></ol></LI&gt; <li>酶溶液<ol><li>加入1毫克/毫升透明质酸酶和胶原酶2.2毫克/毫升的DPBS含0.25微克/毫升的庆大霉素和制霉菌素。准备1毫升这种酶溶液消化2内部肾髓质。该解决方案必须通过过滤灭菌，并存储在50ml Falcon管中。 </li></ol></li></ol> 2。培养原代大鼠内髓集合管（IMCD）细胞所有动物的处理程序进行符合地方和联邦立法。 <ol><li>麻醉大鼠在CO 2（在一个盒子里饱和CO 2）或异氟醚（U-400麻醉机，有限公司，马耳他Univentor;空气流量350-400毫升/分钟2-2.5％异氟醚）。杀头动物出血发白内部较深的外髓之间的边界，允许精确检测。此外，出血孤立的红细胞数量减少到最小。移除肾脏和补充用庆大霉素和制霉菌素（见上文）在冰上的无菌DPBS中把它们洗干净。所有进一步的步骤是无菌洁净工作台条件下进行。 </li><li> DPBS溶液肾脏转移到一个无菌的压缩，以除去过多的液体。取出脂肪肾胶囊用无菌镊子和剪刀。 </li><li>经肾仍然固定在压缩，稍微切的外侧（皮层）的纵向轴线的旁边。确保不完全划破，但把它留在一块，在肾盂连接。 </li><li>用弯剪刀仔细解剖发白的内髓质，被包围玫瑰波光粼粼的外髓。收集他们在DPBS，冰60毫米的培养皿中含有庆大霉素和制霉菌素。 </li><li>一旦所有的髓质分离和收集的冰，减小体积的DPBS的水平，他们只是布满缓冲。使用一个无菌的的剃刀刃切髓质切块53毫米。 </li><li>简单火焰熔化和圆一个无菌巴斯德吸管尖底。确保移液器冷却，然后再继续。 </li><li>组织悬液转移到50毫升猎鹰管含有新鲜配制的无菌酶溶液用透明质酸酶和胶原酶（见1.3.1）用巴斯德吸管圆润。紧紧关上盖子，并在37°C下连续搅拌（220转）一个普通的桌面水浴1.5 - 2小时。 </li><li>研磨的悬浮液，吸液管的混合物向下几次与轮巴斯德吸管（见上文），直到变得均匀混浊的悬浮液。 </li><li>离心悬浮在300 XG 5分钟和16°C。弃上清，悬浮颗粒在DPBS彻底，含有庆大霉素和制霉菌素再次离心。重复此洗涤步骤一次。 </li><li>重悬细胞在完全培养基和种子到c的ulture菜和/或微滴度孔板。 2髓质（1只）的编制将产生足够一个60毫米培养皿中的细胞。 </li><li> 24小时后，洗细胞两次与600 mosmol DMEM，并添加新鲜培养基。在接下来的一周，应清洗细胞的2-3倍（ 图1）。播种后6-8天，髓集合管的细胞，可用于实验。请记住他们在实验开始前24小时，二丁酰环腺苷酸（dbcAMP）和制霉菌素未经孵化。否则AQP2将位于只在质膜。 </li></ol>

<ol>
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作者宣称，他们有没有竞争经济利益。

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。

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			<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 ...

culturing-primary-rat-inner-medullary-collecting-duct-cells

Research

JoVE Journal

Biology

14.7K 次观看.  Max-Delbrück-Center for Molecular Medicine. 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。

视频: Culturing Primary Rat Inner Medullary Collecting Duct Cells

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

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)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

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

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

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 ...

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

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. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

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. ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

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

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

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

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

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

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured 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 ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

大鼠肾上腺皮质细胞的原代培养及非甾体功能的测定

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

多参数灌注对照分离原代大鼠肝细胞

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...