我们要感谢蕾妮L. Pasker和HARPREET K.布拉尔就在这项工作中所描述的协议，专家技术援助。此外，我们要感谢克里斯托弗B. Newgard的师徒在杜克大学和Alan D. ATTIE在威斯康星大学麦迪逊分校，连同他们的实验室的成员，这使得我们的时间的支持，并支持必要的优化描述协议。具体而言，我们感谢汉斯Hohmeier，丹红路和海伦娜温菲尔德在Newgard实验室和玛丽Rabaglia在ATTIE实验室富有成效的讨论和建议。这项工作是由美国国立卫生研究院授予DK080845和青少年糖尿病594支持 研究基金会赠款17-2011-608（以MEK）

<ol>
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作者什么都没有透露。

糖尿病患者预计将影响到世界人口的7.7％的患病率，新颖的研究方法的要求是必须既认识和对待糖尿病18。目前胰岛分离是用于体外实验完善的协议，并已稍作修改11,14,16以前提出。虽然胰岛素分泌的胰岛共同的下游应用，专注于上游的成分，如环磷酸腺苷，可以帮助划定增效胰岛素的产生和分泌的机制。 安乐死的选择必须作出深思熟虑，以确保双方的...

严格的血糖正常的维护是必要的，以防止并发症如神经病，肾病，视网膜病变和，这是不受控制的1型和2型糖尿病1的病理学的所有特点。减少β细胞功能和质量的1型和2型糖尿病扰乱血糖浓度2。而从产生胰岛素的β细胞，受损的β-细胞分泌胰岛素，并在型外围胰岛素信号2型糖尿病的毁灭性损失免疫介导的1型糖尿病患者的结果一起促进高血糖，血脂异常，并增加肝葡萄糖生成，最终导致既损失的β-细胞质量和胰岛素分泌能力从个体β-细胞3。理解在1型和2型糖尿病的进展相关的β-细胞的机制可望产生新的治疗，预防和治疗这些疾病。 体外 TISSUE培养模型，如INS-1和MIN6永生化的β-细胞系，可用于理解特定的β-细胞功能的有用工具。然而，在胰岛内的不同细胞类型的相互作用本身可调节β-细胞的功能。例如，胰高血糖素的旁分泌的影响（解除α-细胞）和生长抑素（从δ-细胞释放）的增加和减少胰岛素的分泌，分别展示了在内分泌反应4芯电池接近的重要性。此外，β-细胞之间的间隙连接增强胰岛素5的释放。此外，虽然进步已在产生胰岛素线路，更好复制的分离胰岛的生理响应葡萄糖（ 例如 ，INS-1衍生十三 ​​分之八百三十二和3分之832细胞系），它们的葡萄糖响应性仍不同于正常大鼠胰岛6,7。此外，这些克隆胰岛素瘤细胞系的反应以胰高血糖素样肽-1（GLP-1）受体激动剂可以从彼此显着不同，以及从正常胰岛6。因此，永生化细胞系可能并不代表用于测定药物对cAMP生成的影响的最佳模式。 与此相反的胰岛来源的细胞系，仅在整体动物模型研究胰岛β细胞功能提供了它自己的一套并发症。其中与内分泌组织工作面临的最大挑战是测量激素的精确浓度释放。具体地说，肝起着重要的作用代谢胰岛素和胰腺的血流直接进入肝脏。因此，血浆胰岛素的测量可能不准确地描绘胰岛素从胰腺自身或不同处理对胰岛素分泌8的速率的影响被分泌的量。另外，胰高血糖素的肾代谢可能限制胰高血糖素输出的可靠度由胰岛α-细胞9。因此，初级分离小鼠胰岛体外实验提供了如何胰岛是响应特定刺激，以补充体内的测量结果更准确的理解。 本协议的小鼠胰岛的分离是通过使用一组数（有轻微的修改，可能有助于提高成功）10,11行之有效的协议。另外，cAMP产生的确定允许直接读出的β-细胞的肠降血糖素应答性。与cAMP的测定，蛋白质含量和胰岛素分泌相结合，也可以从相同的cAMP的样品制备量化，帮助确定在β细胞功能的缺陷是否位于近端或远端的cAMP 10。最后cAMP含量及在本协议中胰岛素分泌的应用程序可以是一个非常强大的工具，为了解医药和饮食成分的影响，其中包括秒，对cAMP和胰岛素分泌。除了 ​​刺激从单独的葡萄糖，其它的化合物可用于测量变化的cAMP和胰岛素分泌10,11。 最后，虽然胰岛素是我们从分离胰岛，其他激素，如胰高血糖素和促生长素抑制素，以及细胞因子，类二十烷酸，和环磷酸腺苷测定的主要激素，也可以测量，无论是由瞬态刺激测定法或通过定量它们的含量在培养基12。最后，虽然本手稿的范围之外，胰岛分离与所述胶原酶分离方法允许对胰岛保存，使得许多其它下游应用可以追求，如胰岛移植，RNA提取用于定量实时PCR或微阵列分析，蛋白隔离蛋白印迹，胰岛嵌入和免疫荧光成像，和[3H] - 胸苷掺入如胰岛细胞REPLI的量度阳离子，其中一些在前面的朱庇特的文章13-16进行了描述。总体而言，继在协议中所述的胰岛分离过程中可提供研究者与开发的疗法，促进药物发现旨在提高胰岛β细胞功能的重要和有用的信息。

<table border="0" cellpadding="0" cellspacing="0" width="502">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Collagenase: Collagenase from Clostridium histolyticum suitable for isolating active islets</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C7657</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Ficoll 400</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>F9378</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hanks Balanced Salt Solution 10X</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td>14065-056</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hepes</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>H3375</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">RPMI 1640 (powder)</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">31800-022</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>A7888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3/0 Silk Suture Thread</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>18020-30</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dumont #5 Forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>11251-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">0.8 mm Forceps&#160;&#160;</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">11050-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Curved Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14061-10</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Vannas-T&#252;bingen Spring Scissors - Straight/Sharp/8.5 cm/5 mm Cutting Edge</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">15003-08</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dissecting Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14002-14</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">5ml BD Luer-Lok Syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309646</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">1ml BD syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309628</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">30 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305106</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">27 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305109</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sharpening Stone</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">29008-01</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-2-2 tribromoethanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">T48402-25G</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-methyl-2-butanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>240486-100mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sodium Chloride (NaCl)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>S9888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Potassium Chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>P3911</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:235px;">Monopotassium Phosphate (KH2PO4)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">P0662</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">Sodium Bicarbonate (NaCHO3)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">S6014</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">CaCl2 *2H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C3881</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">MgSO4 *7H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">M9397</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Penicillin-Streptomycin</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">15140-122</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:235px;">Heat Inactivated Fetal Bovine Serum (H.I. FBS)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:136px;">SH30088.03HI</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">5879-100MG</td>
		</tr>
	</tbody>
</table>

a method for mouse pancreatic islet isolation and intracellular camp determination

不受控制的高血糖是糖尿病的标志，并促进像神经病，肾病，视网膜病和发病率。患有糖尿病，这两种免疫介导1型和肥胖相关的2型，研究目的是区分糖尿病的病理生理和治疗机制的日益普及是至关重要的。 β-细胞朗格汉斯的胰岛负责适当地分泌胰岛素响应于升高的血糖浓度。除了葡萄糖等营养物质，β-细胞被特定的激素也刺激，称为肠促胰岛素，这是从肠道响应对β-细胞受体增产细胞内环磷酸腺苷一顿饭和行为分泌（ cAMP）的。减少β细胞的功能，质量和肠促胰岛素反应是很好理解有助于2型糖尿病的病理生理机制，并且也正在越来越紧密无线第1型糖尿病。本小鼠胰岛分离和cAMP测定的协议可以是一个工具，以帮助界定促进疾病进展和治疗干预，特别是那些由肠降血糖素受体介导的或相关的，通过细胞内cAMP产生的调制作用的受体机制。而仅cAMP的测量将要描述的，所描述的胰岛分离协议创建一个清洁制剂，其还允许在许多其他的下游应用，包括葡萄糖刺激的胰岛素分泌，[3 H] -胸苷掺入，蛋白质丰度，和mRNA的表达。

为了确保隔离在一个高胰岛产量，在协议中所概述的手术技术应密切随访。虽然这里介绍的技术将针对每个实验室有，这将导致一个成功隔离了几个关键步骤。为了使胆总管方便，所以建议的器官被移位到右侧的鼠标（ 图1）。此外，这将使胰腺膨胀以较小的阻力，因为会有较少的重量限制了业务发展。另一个关键步骤，以最大限度地提高胰岛产量胆总管接近奥狄括约肌（ 图2A-...

Watch this Scientific Journal Video about A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination at JoVE.com

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

含量测定采用胰岛分离的小鼠胰岛体外 β细胞功能是在糖尿病病理生理学和治疗学研究的一个重要组成部分。虽然许多下游应用的情况下，该协议特别描述作为必要参数确定的β-细胞功能的细胞内环磷酸腺苷（cAMP）的测定。

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

a-method-for-mouse-pancreatic-islet-isolation-intracellular-camp

Research

JoVE Journal

Biology

65.1K 次观看.  University of Wisconsin-Madison. 含量测定采用胰岛分离的小鼠胰岛体外 β细胞功能是在糖尿病病理生理学和治疗学研究的一个重要组成部分。虽然许多下游应用的情况下，该协议特别描述作为必要参数确定的β-细胞功能的细胞内环磷酸腺苷（cAMP）的测定。 

视频: A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

人类胰岛的分离:第一部分:收集和胰腺组织消化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

科研

生物学

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

人类胰岛的分离:第二部分:人类胰岛的分离纯化及文化

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

小鼠胰岛的朗格汉斯的分离纯化的胶原酶和中性蛋白酶结合使用

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

近红外（NIR）光增加线粒体功能的标志物的表达，在鼠标前庭上皮细胞

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

胰腺胰岛与小鼠分离的简单高效协议

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

小鼠骨髓胰岛移植的一种方法

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

胰岛嵌段石蜡切片

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...