这项工作是支持的，部分由美国国立卫生研究院资助DK060581 R01，R01 DK083583（包括RGM）。

A.所需材料：见表1（试剂）之前的准备<ol><li> HBSS中（P / S）：100毫升10X HBSS + 900毫升H 2 O + 1％青霉素/链霉素（存储在冰箱中或保存在冰中）。 </li><li>在水和20％BSA股票等分并储存于-20℃下</li><li> HBSS中（BSA）：200毫升1X HBSS中（P / S）+ 3毫升20％的牛血清白蛋白（最终BSA的浓度是0.3％） </li><li>小岛培养基：RPMI + 10％FBS + 1％谷氨酰胺+ 1％青霉素/链霉素</li><li> HISTOPAQUE 1100：240毫升1119 HISTOPAQUE 1077 + 200毫升HISTOPAQUE </li><li>切成4-0缝合线（麦克森）：2¾英寸件和存储在一个10厘米的陪替氏培养皿</li><li>工具：从Fine科学工具的钳子弯曲至90°，和文件下来的锯齿状齿所有3双的钳子的。我们的目标是沉闷的牙齿，而不是将其删除。 </li><li>套管管理胶原酶/蛋白酶的混合物：27G½英寸针，30½英寸针，PE50管材切成12英寸件。这两个文件针，一个光滑的圆形到底有没有锋利的边缘。 27G针插入到一端的PE50管一块12英寸。打碎外壳，用一把钳子，打开外壳，取出30G针头的套管¼把每一个时间。用钳子从壳体拔出注射针，在解剖显微镜下，将其插入到PE50管的另一端。确保不纠结你将针头插入PE50管材管。 27G针的结束，你将胶原酶的注射器的连接。 </li><li> ，胶原酶（CI 酶 MA）和中性蛋白酶，（CI 酶 BP）。重组TDE按照生产商的说明。很多具体的分析证书来计算酶的活性浓度。转移共350000胶原蛋白降解活性（CDA）的重组胶原酶单位和10万单位的reconstit中性蛋白酶的uted BP蛋白酶到一个锥形管，并稀释至15ml总在HBSS中（P / S） </li></ol> B.步骤分步协议 1。通货膨胀的胰与胶原蛋白酶/蛋白酶混合<ol><li>安乐死的小鼠颈椎脱位，饱和鼠标躯干，用70％的乙醇，打开腹腔完全从肛门隔膜与任何解剖剪刀和镊子。 </li><li>在解剖显微镜的平台上，将鼠标。 </li><li>拉盲肠和升结肠，他们在你的左边体腔以外的设置。请参阅图1，用于以下步骤的概要。 </li><li>裂片的肝脏对膜片的位置，它们应保持，如果身体腔是开放足够远。肝动脉，门静脉和胆管束，导致肝十二指肠弯钳抓住。另一双弯钳，达到下动脉，静脉和管道不ndle和拉2¾英寸4-0的缝合动脉，静脉，胆管，并把它一次，紧紧地把它接近肝尽可能的。 </li><li>使用弯钳2双，仔细抢十二指肠和找到胆总管连接到十二指肠乳头。使用一堂镊子捅下的乳头附近的胆管，胰腺和结缔组织的权利。把您的镊子迅速通过器官做了一个洞，然后把两个钳，镊子，去抢另一个的2¾英寸4-0缝合线通过，并把它一次，把它的一个小圆圈，但不紧拉。 </li><li>更改为90°钳，和超细维纳斯剪刀的。 90°镊子，小心地捏，拉，直至胆管小肠是绷紧。一个水平贯穿的乳头用剪刀，开放的剪刀刺向进入肠道，然后切割用一个运动。尽量只让Ø东北削减整个乳头乳头不撞出胆管的情况下。 </li><li>虽然仍保持90°肠钳，插入导管进入胆管的胆管长度的一半，然后轻轻拉动缝合紧绕在套管。 </li><li>填充胶原酶/蛋白酶的混合物用2.0毫升的3毫升注射器和插管注入一个缓慢的，恒定的步伐。 </li><li>充气后的胰腺完成后，取出胆管插管从用弯钳和弯曲的弹簧剪刀，删除充气胰腺，开始在降结肠和剪断的结缔组织，你的肠子。从肠道，继续删除胰腺，直到你到达胆管，然后取出胰腺，脾，胃，然后从腹腔的内脏。完成删除剪断了缝合。加入到一个50毫升的试管含有5毫升的HBSS胰腺（P / S）。此管应该在冰上。 </li><li>重复上面的步骤，每个动物多达四种动物。为了达到最佳效果，游离在同一时间只有四个胰腺。通常情况下，前四个是在37°C水浴，膨胀另外四个胰腺。 </li></ol> 2。胰腺分解<ol><li>将灯管中含有50毫升的胰腺，37℃水浴15分钟。 </li><li>轻轻地摇动管和检查组织分离，确保组织分开来容易，然后涡流管的12倍，而你是管的盖子。 </li><li>加入不超过30毫升的HBSS（BSA）（这可以是一个较低的数额，根据需要多少平衡管离心步骤）中，并离心，在290×g离心1分钟。 </li><li>仔细吸液上清液，留下少量的上清液（2-3毫升），以便不破坏细胞沉淀在底部。 </li><li>使用30毫升注射器ATTACHED至14G针，添加不超过10毫升的HBSS（BSA）和吸液悬浮液中向上和向下的2倍。 </li><li>筛选的细胞混合物通过一个塑料滤茶器入50毫升的管，并与另外10毫升HBSS中（BSA）的冲洗。 </li><li>离心330×g离心2分钟。 </li><li>弃去上层清液，反转管排出的吸收纸毛巾，持续1分钟。 </li><li>每个管重悬在10毫升冷1100 HISTOPAQUE。 </li><li>叠加HISTOPAQUE用10ml HBSS（BSA），并在900×g离心18分钟，离心。 </li><li>取出整个20毫升的上清液，使用一个大孔25毫升移液管的上清液，并通过70微米的过滤器通过倒置。 </li><li>冲洗胰岛到一个10厘米的陪替氏培养皿中，通过过滤器，同时保持它的右侧在菜通过移液10毫升胰岛媒体。 </li><li>在37°C，5％CO 2组织培养孵化器文化的小岛，直到准备进行试验。通常情况下，胰岛手工采摘和计算之前experimentation。 </li></ol> 3。代表性的成果胰岛的产量，形态和质量的常规参数来判断成功的胰岛分离。在我们的手中，平均C57BL / 6小鼠胰岛的产量是使用该协议的（见表2）150-250小岛的范围内，得到优化的Σ型XI型胶原酶的数据具有可比性。然而，产率可以有所不同，这取决于使用的小鼠品系和鼠标的年龄。大部分的动物，我们采用的是在8-16周龄的范围内，该协议在多个小鼠品系进行了测试，其中包括C57BL / 6（180-250小岛/鼠标），CD1（200-300小岛/鼠标），129 / B6（200-300小岛/鼠标），BLKS-db/db（120-200小岛/鼠标），NOD（10周龄100-150小岛/鼠标），NOD-SCID（100-150小岛/鼠标）。典型的胰岛形态如图2所示，在其中胰岛有一个圆形的长椭圆形的形状具有相对均匀的尺寸（虽然大小单位一的ormity可以从应变应变）。 图3显示了典型的分析，葡萄糖刺激胰岛素分泌的C57BL / 6小鼠胰岛的分离用纯化的TDES与Σ型XI胶原酶。通常情况下，一个高质量的的胰岛制备结果在25mM葡萄糖是6-20倍大于观察到在2.5 mM葡萄糖胰岛素刺激。其他应用程序也可用于测试的质量分离胰岛，其中包括使用表面培灌技术，Ca 2 +的成像（与Fura2），和逆转录PCR，其中包括11。 <img src="/files/ftp_upload/4137/4137fig1.jpg" alt="图1" /> 图1。概要的胆管插管。 <img src="/files/ftp_upload/4137/4137fig2.jpg" alt="图2" /> 图2。典型的外观C57BL / 6小岛在24小时以下的隔离。一个在图像被收购倒置显微镜在40倍的放大倍率。 <img src="/files/ftp_upload/4137/4137fig3.jpg" alt="图3" /> 图3。葡萄糖-刺激的胰岛素分泌的C57BL / 6小鼠胰岛在 24小时以下隔离，100的胰岛培养在12孔盘1小时，在37℃，HEPES缓冲的Krebs-Ringer溶液含有2.5mM葡萄糖。胰岛，然后转移到一个额外的小时，此后，收集上清液用于胰岛素测量在2.5 mM葡萄糖的Krebs-Ringer HEPES使用双位点免疫特异性的酶联免疫吸附试验（ELISA）（晶体化学）。相同的胰岛，随后将其转移到HEPES缓冲的Krebs-Ringer溶液含有25mM葡萄糖，胰岛素释放到培养基中孵育1小时后，用ELISA法测定。

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NDS，SAT，RGM没有透露。作者的AGB采用VitaCyte。作者RCM拥有所有权在VitaCyte的兴趣。

在这个协议中，我们已经描述了我们的程序插管术，胶原酶灌注，分离的小鼠胰腺与胰岛分离的目标。从技术上讲，我们的方法，一旦掌握了，应该允许在4分钟内实施安乐死的动物的胰腺隔离，1小时应该从一个单一的动物在隔离的小岛。该技术的快速性允许我们分离胰岛从最多至12只小鼠，在一个典型的拉伸，从而导致高达3000胰岛隔离。 我们的协议与其它协议不同，胶原酶制剂的使用原油，设有纯化的蛋白酶，CI 酶胶原酶MA和CI 酶 BP蛋白酶的使用。 TDE制剂市售的一致性的变化，需要每个特定单独测试和优化一般使用之前。这很多很多的变化，促使我们考虑使用纯化田间的TDES从VitaCyte。这些高度纯化的TDES其特征在于由多个敏感的荧光标记底物测定的使用方法，包括。的荧光CDA检测是更加敏感，不同的分子形式的胶原酶降解天然胶原蛋白具有不同的动力学。历史的多肽底物检测提供胶原酶的评估是不完整的。特征胶原酶多种方法，确保批次间更大的酶的活性。 根据我们的内部测试，使用各种市售胶原酶制剂，我们发现，纯化的酶组合，产生了很多很多的重复的结果。在此应用程序中使用的高纯度和严格的特点TDES的主要优点是一次最佳的酶组成的定义，很多很多的性能的一致性是有保证的。这种一致性消除劳动力密集的过程通常需要很多资质原油或丰富胶原酶产品。纯化的TDES也使额外的修改，以提高产量和质量的胰岛细胞恢复从不同的小鼠品系的组成。报告最佳的酶分离胰岛的组合物可更有效地从不同品系的小鼠连通。这反过来又导致更有效的使用资源，提高灵活性，在实验设计中。 有很多关键的步骤，可以影响胰岛的隔离使用我们的协议的结果。第一个是需要的酶制剂的灌注过程中，以实现有效的通货膨胀的胰腺。重要的是要启动注射器上的通胀温和的力量，和增加胰腺充气力。移动的肠子和解除通胀，使的胶原酶灌注整个器官，胰腺是有帮助的。另一个关键步骤是一甩头管竣工后的力ŕ孵化，在37°C（2.2）。我们发现，难防摇胰腺管帽引起的胰岛细胞的分裂，但没有任何形式的机械分离这一步，胰岛产量大幅下降。此外，还必须在与注射器（步骤2.5）的解离步骤，一个中等级别的力被施加在与柱塞的向上和向下运动期间，这之前的滤波步骤（2.6），以确保有足够的组织分离，其中很少组织应保留在滤茶器过滤器。最后，最后一个步骤仔细遵循的是在整个20毫升的HISTOPAQUE分离胰岛细胞的愿望。虽然的小岛是典型的在界面上的HISTOPAQUE和HBSS，我们已经发现，我们失去了小岛时，我们尝试只删除接口，相反，我们现在可以删除整个20毫升，用大口径25毫升的移液管。我们添加了过滤，以消除需要通过倒置70微米的过滤器（2.11）到centrifuge的小岛，反复洗去HISTOPAQUE。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td></td><td> 目录＃ </td></tr><tr><td> 10X HBSS </td><td> Life Technologies公司</td><td> 14065 </td><td></td></tr><tr><td> RPMI </td><td> Fisher Scientific则</td><td> 10-040 </td><td></td></tr><tr><td> BSA </td><td> Sigma Aldrich公司</td><td>任何</td><td></td></tr><tr><td> HISTOPAQUE 1077 </td><td> Sigma Aldrich公司</td><td> 10771 </td><td></td></tr><tr><td> HISTOPAQUE 1119 </td><td> Sigma Aldrich公司</td><td> 11191 </td><td></td></tr><tr><td> 4-0缝合线100码卷</td><td>麦克森</td><td> 451521 </td><td></td></tr><tr><td> 14克钝端针</td><td> Fisher Scientific则</td><td> 14-8216M </td><td></td></tr><tr><td>Vannas超细Sciss </td><td> Fisher Scientific则</td><td> 501778 </td><td></td></tr><tr><td>曲春风似剪刀</td><td> Fisher Scientific则</td><td> 14127 </td><td></td></tr><tr><td>弯钳（2） </td><td> Fisher Scientific则</td><td> 15914G </td><td></td></tr><tr><td>弯钳（90°） </td><td>精细科学工具</td><td> 11052-10 </td><td></td></tr><tr><td> 27G 1/2寸毫针</td><td> Fisher Scientific则</td><td> 305109 </td><td></td></tr><tr><td> 30G的1/2寸毫针</td><td> Fisher Scientific则</td><td> 305106 </td><td></td></tr><tr><td> PE管材</td><td> Becton Dickinson公司</td><td> 427411 </td><td></td></tr><tr><td>胶原酶MA </td><td> Vitacyte，LLC </td><td> 001-2030 </td><td></td></tr><tr><td>乙P蛋白酶</td><td> Vitacyte，LLC </td><td> 003-1000 </td><td></td></tr><tr><td> 3毫升的注射器</td><td> Fisher Scientific则</td><td> 309585 </td><td></td></tr><tr><td> 30毫升的注射器</td><td> Fisher Scientific则</td><td> 309650 </td><td></td></tr><tr><td> 70微米的过滤器</td><td> Fisher Scientific则</td><td> 352350 </td><td></td></tr><tr><td> 10平方厘米培养皿</td><td> Fisher Scientific则</td><td> 351005 </td><td></td></tr><tr><td>塑料滤茶器</td><td>任何厨房供应</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 表1。试剂和仪器 </td></tr><tr><td></td><td></td><td></td><td><table border="1" cellspacing="0" cellpadding="0" width="510"><tbody><tr><td> Σ型XI （l010M8618） </td><td> MA（100615） BP（110329） </td><td> MA（081104） BP（100823） </td><td> MA（091211） BP（101109） </td></tr><tr><td> 216（±74） </td><td> 260（±45） </td><td> 157（±37） </td><td> 200（±4） </td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td> 第2表平均的胰岛收益率（±SD）使用不同的的TDE手* *数据代表平均至少有10只小鼠的胰岛产量。地段括号中的数字</td></tr></tbody></table>

mouse islet of langerhans isolation using a combination of purified collagenase and neutral protease

β细胞的基因表达和功能的体外正视转移的询问多年来的研究孤立的啮齿类动物胰岛细胞从啮齿动物致瘤细胞株的研究。主要的小岛提供了独特的优势，他们更忠实地反映细胞内的信号转导通路的生物学特性及分泌反应。而胰岛分离的方法组织解离酶（TDE）的筹备工作已经确立1-4的许多实验室，在任何给定的啮齿动物应变的的胰岛产量和质量的一致性的变化限制的范围和主要胰岛研究的可行性。这些变化经常会出现作为一个结果，部分纯化的粗胰岛的分离过程中使用的TDES; TDES经常表现出特定对特定活动的变化，并常常需要调整所用的酶的剂量。纯化TDES啮齿动物细胞隔离5，6，少数报告</suP&gt;，但这种做法并不普遍，尽管日常使用和纯化TDES人胰岛隔离的优势。在与VitaCyte，LLC（印第安纳波利斯，IN）的协作，我们开发了一种修饰的小鼠的胰岛分离协议基于Gotoh琴桥7，8，在该TDES灌注直接进入胰管小鼠，随后粗组织分馏通过所述的一个HISTOPAQUE梯度9纯化的胰岛细胞，并进行隔离。甲我们的协议的显着差异是纯化的胶原酶（CI 酶马萨诸塞州）和中性蛋白酶（CI 酶 BP）的组合使用。胶原酶，其特征在于通过使用6荧光胶原降解活性（CDA）的检测，利用荧光标记的可溶性小腿皮肤纤维作为基板6。此衬底是更预测组织基质中的胶原降解的动力学，因为它依赖于天然胶原作为基板。该蛋白酶为ch的aracterized与一个敏感的荧光动力学分析10。利用这些改进的检测与传统的生化分析，使TDE制造更一致，批次间的一致性来提高性能。在这里描述的协议进行了优化，以获得最大的的胰岛产量和最佳胰岛形态C57BL / 6小鼠。胶原酶和中性蛋白酶在此协议的发展过程中，有几个组合进行了评估在不同浓度，胶原酶：中性蛋白酶35:10的最终比例-Σ型XI代表酶的性能相媲美。因为在不同品系的小鼠和大鼠胰岛产量平均显着的变化已有报道，额外的修改的TDE组成的，应以提高产量和质量的不同品种和品系的胰岛细胞恢复。

Watch this Scientific Journal Video about Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease at JoVE.com

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

小鼠胰岛分离的详细描述描述原位胰腺导管插管和灌注纯化的胶原酶和中性蛋白酶的组合中使用的技术。

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 ...

mouse-islet-langerhans-isolation-using-combination-purified

Research

JoVE Journal

Biology

43.2K 次观看.  Indiana University School of Medicine. 小鼠胰岛分离的详细描述描述原位胰腺导管插管和灌注纯化的胶原酶和中性蛋白酶的组合中使用的技术。

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

Murine Pancreatic Islet Isolation

小鼠胰岛隔离

科研

生物学

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 ...

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

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

分离小鼠原代滋养细胞及滋养细胞侵袭实验

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

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 ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

人肝细胞通过一个两步胶原酶灌注过程隔离

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

确定膜蛋白拓扑使用荧光蛋白酶保护（FPP）

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

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 ...