这项研究是由国家卫生部授予R01HL086939（NCC）研究院的支持。

在这个协议中，我们展示了一个孤立的，通风，灌注小鼠肺准备以前已使用量化肺栓塞和慢性缺氧对搏动性肺血管压力流量关系的影响（Tuchscherer，韦伯斯特，Chesler，2006年; Tuchscherer等，2007）。简言之，小鼠肺手术从周围组织中分离出，放置在一个加热室（IL - 1，哈佛大学器械，Holliston，马）和通风（通气控制模块（VC​​M）- R的定时器计数器模块（TCM），哈佛大学器械）。肺血管灌注加热的RPMI 1640细胞培养液中，用3.5％聚蔗糖，使用注射泵（科尔 - 帕默，美国Vernon Hills，IL）产生源源不断的波形或一个高频振荡泵（玻色 - 电磁力，伊甸园大草原，MN）在与注射泵创建搏动性肺血管流波形并联。压力传感器（P75，哈佛仪器）测量瞬时肺动脉压（PAP）和左心房压力（LAP）。瞬时流量（Q）是衡量行流量计（跨音速系统公司，美国纽约州伊萨卡）。脉动压力 - 流的关系是来自这些测量，提供洞察到肺血管的生理和病理及右心室后负荷。 1。设备： <ol><li>肺隔离设置，包括鼠标呼吸机</li><li>数据采集​​系统和计算机与LabVIEW程序</li><li>两个压力传感器和流量计灌流流</li><li>压力传感器和流量计（pnemotachometer）气道流</li><li>高频振荡泵和计算机用Win测试程序</li><li>臂/变焦镜，灯</li><li> IL - 1系统具有高输出泵的暖气浴</li></ol> 2。准备的IL - 1系统<ol><li>蒸馏水加热至37 ° C的加热浴的IL - 1系统的内部传阅。 </li><li>所有泵，传感器和IL - 1套管连接，通过清洁管子和所有油管刷新蒸馏水加热至37 ° C。气泡，这可能会进入肺部，引起水肿旅游，必须拆除。振荡流泵，流量传感器，流量传感器的油管肺动脉导管，用1％PBS冲洗。 </li><li> P75压力传感器归零，关闭阀门的导管，打开阀门的气氛，然后推PLUGSYS放大器自动调零按钮。然后，到大气中的阀门关闭，打开阀门的导管。 </li><li> IL - 1系统的通风途径的多孔陶瓷片湿提供湿度。 </li></ol> 3。解决方案<ol><li>准备量聚蔗糖的RPMI解决方案和无菌过滤介质3.5％。过滤介质，确保没有大的颗粒可能无意中栓塞肺。用无菌媒体也减少了突发发展中国家在肺水肿的可能性。每个实验试验手术用RPMI一个60毫升注射器，用RPMI填写一个10毫升注射器。在37℃水浴加热的灌流。 </li><li>准备肝素鼠标（近似）500IU/100g体重1毫升。肝素盐158IU/mg。为1〜25克鼠标，用1ml PBS解决方案，在一个小的离心管中混合1.25毫克肝素盐。 </li></ol> 4。通风一个滑鼠指标<ol><li>后腹腔注射150毫克到老鼠的每公斤溶液中苯巴比妥，确保深麻醉，一爪子上执行硬捏。如果没有反应，准备手术鼠标寄希望于它的前爪corkboard稳定。注：整个实验过程中麻醉深度监测，仔细观察动物。爪子和频繁的切口引脚作为伤害性刺激，缺乏应对证实，动物仍然在手术麻醉平面。 </li><li>喷雾用95％酒精湿毛皮和使用直钳抓住颈部皮肤的胸部。切1厘米的皮肤在使用直剪刀开放。 </li><li>一旦暴露颈部内侧，除去所有的白色腺体组织和浅肌层，寻找食道和气管。从组织分离的食管和气管两侧和后部。 </li><li>下插入气管的小弯钳和缝合对方抢到一块。根据气管拉缝合，并配合一个松散的外科医生结。不要收紧缝合或喜结良缘。 </li><li>切一个小角度，在气管与小剪刀“V”，不切断所有通过气管。软木板和鼠标移动到加热的IL - 1系统。使用两个钝钳，抢气管插管和气管下方的“V”。然后，滑入气管插管泽EA通过的“V”开幕。拧紧气管周围的缝合和气管插管，以确保里面的气管插管。喜结良缘。 </li><li>开始与室内空气的通风（50％的灵感，90次/ min，深吸气，）。 </li></ol> 5。灌流一个通风的鼠标 5.1。进入右心室注入肝素<ol><li>喷雾酒精鼠标胸部再次重新弄湿皮草。以上胸部的肋骨，用直钳和直剪上删除所有的皮肤。沿胸骨向上切。电梯在每边的皮肤，然后切开皮肤下面较低的肋骨。 </li><li>抓住在胸骨用镊子底部剑突过程中，切孔进入隔膜采用了直板的剪刀。抓住镊子的隔膜，从肋骨。 </li><li>再用镊子抓住剑突（左手）和使用的球头，直角剪刀切断了通过肋骨的胸骨和，小心不要切肺，心脏或血管（使用球头剪刀来引导你）。将有血，但只要是剪刀的尖端对胸骨，心脏和肺部不会被削减。 </li><li>抓住左侧肋骨和肋骨必要暴露心脏剪去。慢慢地注入0.1毫升的肝素液的右心室。这一步很重要，以防止血液凝块，在肺，损伤内皮细胞，并会损害灌注。必须注射肝素，而心脏还在跳动。 </li></ol> 5.2。 Cannulate主肺动脉<ol><li>切去其余的肋骨（左侧和右侧）使用回（四舍五入）结束镊子轻轻推肺部远离肋壁。敛肺，自己会损害娇嫩的组织。剪刀的技巧和肺组织之间的偶然接触也会造成损害。 </li><li>显微镜移动到超过肺癌的地方。切去心脏上方的腺体和脂肪组织。使用镊子把它拉离的动脉和静脉，然后用弹簧剪刀削减，而组织张力。 </li><li>使用另一个镊子钝瓢，从心顶部左侧下左心房/心室下的主动脉和肺动脉（PA）的镊子的尖端。仔细做到这一点不应该有抵抗镊子。使用生硬的镊子降低，不小心刺破肺动脉或主动脉的可能性。 </li><li>一旦镊子尖下的主动脉和PA，抢一块缝合，转危为安。打结宽松的外科医生。不要收紧缝合或喜结良缘。 </li><li>可选：使用直角剪刀去除身体的下半部分。减少通过肋骨和脊柱;切割主动脉和腔静脉会造成大量的血液流向 - 使用棉条，以坚定的流量。放置在一个袋子处置。 </li><li>总理PA导管与4毫升的RPMI 10 ml注射器从。仔细检查所有的管子无气泡。灌注肺，用蒸馏水或气泡会导致水肿。 </li><li>剪下右心室游离壁的小凹口，插入PA导管，旨在向下和向右。导管的尖端应通过透明墙的PA可见。注入少量的RPMI确认您的位置。周围的导管，主动脉和PA拧紧缝合，并喜结良缘。请注意，在这一点上，主动脉也追平，所以实际灌注不应该开始直到左心房是空心的。 </li></ol> 5.3。 Cannulate的左心房<ol><li>切了一个档次，降低左心室的一部分，和惰性的左心房（LA）的套管，旨在向上。轻微的压力，可能需要打开这个方向的二尖瓣。在正确的位置，导管尖端漏网之鱼和安全无需缝合。 </li></ol> 5.4。开始灌注<ol><li>手动注入0.3毫升每分钟的RPMI直到RMPI是显而易见的，在流出管（粉ISH在色彩对比明确PBS），从10毫升注射器。如果没有流流出管，重新定位的LA套管。如果没有流出可以得到重新定位，检查肺动脉泄漏。在肺动脉泄漏或撕裂，无法修复;这是导致中止实验。 </li><li> 60毫升注射器连接PA的导管通过IL - 1系统，并开始为1ml/min灌流输液，检查泄漏和确保肺部变成白色，这说明的RPMI是取代在肺部的血液。两分钟的慢血流灌注。 </li></ol> 6。测量脉动和稳定的肺动脉高压流量关系<ol><li>脉动流的研究，第一组的振荡泵活塞位​​移WinTest根据以前的实验方案针对每一个频率所需的水平。由于变性，肺的结构和力学的位移必须为每个鼠标调整。源源不断设置到所需的程度。打开阀门的振荡泵，并开始运行前的振荡流配置文件（Wintest程序）记录数据。打开数据文件，并绘制在Excel（Q）的实验流程。调整在每个频率的振荡泵活塞位​​移（Wintest方案）使Q MAX和Q民需要。 </li><li>稳流试验，关闭阀门的振荡泵。如果此阀处于打开状态，振荡泵作为一个电容器，抑制的变化到另一个，从一个流量。收集至少10秒的数据，在每个流量或直到PA压力不超过5％。 </li><li>搏动性或稳定的流量测量，在数据收集前的恒压老通风。数据收集后立即恢复通风。 </li><li>观看的RPMI在气道管，这是水肿的证据，并中止试验的原因。另外，不要让我们的RPMI达到气道压力或流量传感器，因为它会损坏传感器。 </li></ol> 7。代表性的成果： 代表稳定的结果： 设置隔离肺实验者有能力独立控制，不仅肺流量Q，但也气道压力P空气和左心房压力圈。这是有利的，因为Q，P 空气和LAP影响肺血管，导致肺动脉压PAP的。另一个好处是，得到的结果是独立于交感神经系统的音1，卷的状态，和麻醉2。 在Q阶梯变化引起的空气固定的p和固定或不同的LAP PAP的变化如图1所示。请注意，在孤立肺准备，LAP的导管通常连接管，指示物的容器灌流。有了这个油管，LAP是问，由于Poiseuille流线性依赖。但是，可以手动调整的出口和废物容器的高度，以提供一个恒定的，非零圈或油管可以去掉，让零LAP的独立是问代表脉动结果： 虽然可以用此系统生成任意脉动流波形，我们通常会产生流动的形式：Q = 3 + 2罪（2 fπt）毫升/分钟的 F = 1，2，5，10，15和20赫兹的频率评估肺血管（图2：顶部面板）的线性阻抗。从PAP，搭接和Q测量，计算肺血管阻抗值（Z）和相位（θ）第一每个施加正弦流量频率分解成一系列ΔP= PAP - LAP和Q的一个完整的正弦周期使用傅立叶变换的正弦谐波。压力比改造流向变换产生的肺vascualar阻抗，PVZ = FFT（△P）/ FFT（问），幅度Z和相位θ。输入阻抗Z 0的特性阻抗Z C，和波反射ř W指数，计算出阻抗值。 Z 0，特别是从Z计算零谐波（F = 0赫兹）的平均以上的所有频率，Z C的Z之间的第一次最低（5赫兹）和20 Hz的平均计算，和ř W是计算（Z 0 - Z C）/（Z + Z C）3。 <img src="/files/ftp_upload/2690/2690fig1.jpg" alt="图1" /> 图1稳流波形（上排）和由此产生的压力（第二行：PAP，P 空气 ，LAP）与圈和P 空气不同的组合功能的时间。最后一行显示与问：人民行动党在（A）和（B）与Q的单圈成绩的增加和下降，因为流出管到位。 （三）使LAP是恒定的，并问：独立，在（D）和（五）出口管的高度为手动调节，这样的LAP较高， 但独立于问：P空气，无论是在油管被删除吸气末（A，C，D）或呼气末（B，E）的压力。 <img src="/files/ftp_upload/2690/2690fig2.jpg" alt="图2" /> 图2脉动流波形问（顶部面板）和压力（底部面板：PAP，LAP和P 空气 ）作为时间的函数。 PVZ从这些搏动性肺压力 - 流的关系，可以计算的，它反映了总的右心室后负荷。

<ol>
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在手术的关键步骤这是关键，护理是采取切割时从肺部肋骨。必须充分暴露肺和不羁周围组织在通货膨胀，但不是在隔离的过程中损坏。可用于使用一个平面物体如镊子后端举行胸壁肺剪刀削减，以便有一个清晰的路径。另一个关键的一步是缝合肺动脉和主动脉周围的位置。使用钝直镊子将降低肺动脉穿刺的风险。手术过程中的最后关键的一步是插管的位置。如果套管以上的飞...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>1 ml syringe</td>
<td>&nbsp;</td>
<td>Fisher Sci</td>
<td>14-829-10F</td>
<td>&nbsp;</td>
<tr>
<td>10 ml syringe</td>
<td>&nbsp;</td>
<td>Fisher Sci</td>
<td>14-823-2A </td>
<td>&nbsp;</td>
<tr>
<td>60 ml syringe</td>
<td>&nbsp;</td>
<td>Fisher Sci</td>
<td>13-689-8 </td>
<td>&nbsp;</td>
<tr>
<td>RPMI with GLN 6/PK</td>
<td>&nbsp;</td>
<td>Fisher Sci</td>
<td>MT10040CV </td>
<td>&nbsp;</td>
<tr>
<td>Bottle Top Filters</td>
<td>&nbsp;</td>
<td>Fisher Sci</td>
<td>09-761-57</td>
<td>&nbsp;</td>
<tr>
<td>Ficoll PM 70</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F2878-100g</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Y27632</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>Y0503</td>
<td>&nbsp;</td>
<tr>
<td>Angled Ball Iris scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14109-09</td>
<td>&nbsp;</td>
<tr>
<td>Vannas Spring Scissors - 4mm Blades</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15018-10</td>
<td>&nbsp;</td>
<tr>
<td>Fine Iris Scissors - straight</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14106-09</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5/45 Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11251-35</td>
<td>&nbsp;</td>
<tr>
<td>Dumont Medical Biology Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11254-20</td>
<td>&nbsp;</td>
<tr>
<td>Lauda E100 ECO-line 003 </td>
<td>&nbsp;</td>
<td>VWR</td>
<td>Comparable to Lauda-Brinkmann E-103, 62400-922</td>
<td>&nbsp;</td>
<tr>
<td>IL-1 Isolated perfused mouse lung system </td>
<td>&nbsp;</td>
<td>Harvard Apparatus </td>
<td>739904 </td>
<td>&nbsp;</td>
<tr>
<td>Blood Pressure Transducer P75 for PLUGSYS Module</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>730020</td>
<td>&nbsp;</td>
<tr>
<td>TS410 Flow Modules</td>
<td>&nbsp;</td>
<td>Transonic </td>
<td>TS410</td>
<td>&nbsp;</td>
<tr>
<td>ME 4 PXN Precision PXN Inline Flowsensors</td>
<td>&nbsp;</td>
<td>Transonic</td>
<td>ME 4 PXN</td>
<td>&nbsp;</td>
<tr>
<td>Cole-Parmer Multi-Syringe Pumps</td>
<td>&nbsp;</td>
<td>Cole-Parmer </td>
<td>EW-74900-20</td>
<td>&nbsp;</td>
<tr>
<td>Nembutal 50MG/ML 20ML Vial</td>
<td>&nbsp;</td>
<td>Amatheon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

characterization of the isolated, ventilated, and instrumented mouse lung perfused with pulsatile flow

隔离，通风，允许稳定和脉动的肺血管压力 - 流的关系，对肺动脉流量，流速波形，气道压力和左心房压力独立控制测量仪表小鼠肺癌准备。基于多点，稳定的压力流量曲线计算肺血管阻力，肺血管阻抗是搏动性的压力 - 流的频率范围内获得的曲线计算。由于目前临床上公认的，阻抗比电阻右心室后负荷的一个优越的措施，因为它包含了血管顺应性的影响，这是不容忽视的，尤其是在肺循环。 “三个代表”重要指标的阻抗-零赫兹阻抗Z 0，特性阻抗Z C，和波反射ř W指数-提供洞察到远端动脉横截面积流量，近端动脉僵硬度和上下游的阻抗不匹配，分别为。在隔离所得的全部结果，通风和灌注肺是独立于交感神经系统的音，卷状态和麻醉的效果。我们已经用这种技术来量化肺栓塞和慢性缺氧电阻和阻抗的影响，并区分行动网站（即近端与远端）血管活性药物和使用 Z C的压力依赖的疾病。此外，当这些技术与基因工程菌对小鼠的肺部用于，分子水平上对肺血管的结构和功能缺陷的影响，可以决定的。

Watch this Scientific Journal Video about Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow at JoVE.com

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

以下协议概述的隔离，通风和稽核小鼠肺组织措施稳定或搏动性肺血管压力 - 流的关系，以量化的血流量，气流，气道的变化和血管的变化，右心室后负荷的影响的过程。

表征隔离，通风，和仪表小鼠肺灌注与脉动流

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

characterization-isolated-ventilated-instrumented-mouse-lung-perfused

Microsoft Bing

Research

JoVE Journal

Biology

16.1K 次观看.  University of Wisconsin – Madison. 以下协议概述的隔离，通风和稽核小鼠肺组织措施稳定或搏动性肺血管压力 - 流的关系，以量化的血流量，气流，气道的变化和血管的变化，右心室后负荷的影响的过程。

视频: Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

科研

生物学

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

微型机械的肺组织利用原子力显微镜表征

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett&#8217;s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

An Immunofluorescent Method for Characterization of Barrett&amp;#8217;s Esophagus Cells

There is a discernible need for improved information on molecular drivers of Barrett&#8217;s Esophagus. Immunofluorescent staining is a useful technique for understanding the effects of cell signaling on cell morphology. We present a simple, effective protocol for the use of immunofluorescent staining to assess therapeutic treatment in Barrett&#8217;s Esophagus cells.

免疫荧光法Barrett食管细胞的表征

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

表型鼠标肺功能<em&gt;在体内</em&gt;与肺弥散功能

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.