该研究是由美国国立卫生研究院HL75503以KP支持。

对动物进行的所有实验均批准的田纳西大学健康科学中心大学的机构动物护理和使用委员会。 1，油管灌注大鼠肺制剂<ol><li>准备一个管道系统用聚乙烯管（＃18），用于血液灌注， 如图1连接的压力传感器（P23XL），并放置在显微镜载物台上的管件。 </li><li>设定水浴温度为37°C。 </li><li>连接肺入口，并与聚乙烯管暂时出口。 </li></ol><img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51552/51552fig1highres.jpg" width="500" /> 图1。血液灌注管 。该图显示了用于通过隔离肺制剂血液循环管路的设置。也表明是相关联的组件，通过该管子我s路由。心脏和肺的示意图包括显示管和肺动脉（PA）之间的连接部位，和左心房（LA）套管（蓝色虚线）。 2，制备离体大鼠肺<ol><li>麻醉大鼠（雄性Sprague-Dawley大鼠; 250-300克）用氯胺酮（80〜100毫克/千克），并用甲苯噻嗪（5-10毫克/千克）。后确保手术的麻醉平面，将动物仰卧位置。 </li><li>进行气管切开，插入气管插管（PE-90），并与手术缝合固定。 </li><li>通过心脏穿刺（21克蝴蝶针）注入肝素（100-200 U）到心脏，等待60秒，抽血血（〜12-15毫升血）。 </li><li>准备两个套管（聚乙烯管，直径3毫米，4厘米长，扩口的一端），并填充生理盐水。 </li><li>进行开胸手术。做一个切口（3毫米），在右心室和套管的扩口端滑入朝向肺切口动脉。固定套管肺动脉与手术缝合线。 </li><li>切割（3毫米），左心室心尖和第二套管的扩口端滑入切口。引导导管进入左心房。固定插管至左心室与脐带（2毫米宽）。 </li><li>解剖任何结缔组织，并与所连接的插管取出肺和心脏在一起。将肺和心脏的培养皿。重新定位的肺，使膈面在上面。这三个套管应朝向同一方向。 </li><li>放置在能够在XY方向上进行操作的一个阶段的肺制剂。附带显微镜任何阶段都可以进行修改，以保持管件或内置，如果需要一个特制的阶段。 </li></ol> 3，血液离体大鼠肺灌注<ol><li>混合放血血用等体积的白蛋白（5％）溶液，并添加到储存器。启动蠕动泵和设置流程R吃了14毫升/分钟。使血液填充管上。 </li><li>与分流开，取出肺临时进出水口接头。附加肺动脉和左心房插管至肺入口和出口，分别。确保没有气泡在管。 </li><li>气管插管连接到第三压力传感器（P23XL）。通过气管插管肺膨胀与30％氧气和压力保持5厘米高2 O。 </li><li>关闭分流钳，开始灌注肺。维持肺动脉和〜分别为10 3厘米H 2 O的左心房压力。 </li></ol> 4，准备对肺微血管灌注<ol><li>提高对上面的肺和安全水平的导管延伸管的后端。 </li><li>通过插入一个长40厘米PE10管材成约30厘米长的PE90管长约30厘米准备输液导管。在PE10管的外露端连接到一根针（30克）在上亦有一个注射器（1毫升）。 </li><li>插入导管组合成延伸管的凸起尾部。通过左心房和进入肺引导导管组合，直到遇到阻力。然后，再独自推PE10导管进入肺部，直到它遇到阻力。需要注意的是用力过大，同时插入导管会损害肺。 </li><li>填充1毫升注射器用林格氏溶液和附加到一个注射器泵。设置输液速度以10微升/分钟。 </li><li>相比于肺的其余部分注射部位会变得苍白。 </li><li>润肺用生理盐水，盖上保鲜膜，有孔大到足以离开输液部位发现的肺。 </li><li>棉签上的O形环（定制）的底部部分旋塞润滑脂，并放置在玻璃盖玻片（＃1.5，直径为22mm）上的O形环的底部。轻轻地用在输注部位的盖玻片放置O型圈。固定O形环与标准试管方荣景DER。盖玻片/ O型圈组合不变形的肺泡结构，它的下面，如最近报道15。 </li><li>位置上方的O形环的荧光显微镜的物镜（20X），并集中于肺表面。 </li></ol> 5，成像荧光葡聚糖过境微血管<ol><li>准备显微镜成像FITC荧光。 </li><li>选择要成像的区域，并利用图像采集软件在1/minute的速率采集图像。 </li><li>使用注射泵开始输注FITC-葡聚糖20 KD（0.5毫克/毫升）的。荧光血管会逐渐增加，达到最大值。继续输注60分钟。 </li><li>然后切换到林格氏输液洗掉管腔荧光。输液维持超过10分钟。在洗涤过的继续图像采集在1/min极限。 </li></ol> 6，图像分析<ol><li>打开图像采集文件。 </li><li>地方区域利息在图像帧中的微血管。 </li><li>播放的图像文件的帧接一帧并记录在感兴趣的所有帧的每个区域的荧光强度。 </li><li>绘制荧光强度的变化的利息作为时间的函数的每个区域。 </li><li>量化的最大荧光强度和冲刷10分钟后残留的荧光强度。 </li><li>计算最大值之比，以在每个感兴趣的区域残留的荧光强度，以获得与所关注该区域相关联的微血管的渗透性指数。 </li></ol>

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作者什么都没有透露。

孤立的血液灌注肺标本加上实时成像提供了确定在单肺微血管通透性改变一个简单的工具。我们采用该方法来定义响应于LPS输注渗透性的变化。我们的数据清楚地表明，脂多糖注射引起的增加微血管通透性。此外，数据还表明，LPS诱导的通透性的改变是相似的在两个小静脉和毛细血管。因此，这种技术的主要优点是定义单个微血管的泄漏，以及一个全局血管网络的能力。鉴于这种技术也产生全球?...

在肺微血管通透性增加导致肺泡水肿和损害气体交换的发展，是急性肺损伤（ALI）1-3的一大特点。因此，血管通透性的估计是在确定肺损伤，并提出治疗措施疗效的程度很重要。重量分析诸如血液中的游离肺湿重与干重比和微血管过滤系数被广泛使用的方法来估算渗透性4,5。其它方法包括定量肺组织6-8中的放射性或荧光探针的保留。然而，上述方法需要肺组织样品的postexperiment加工朝向阐明的渗透率数据。此外，由于一动物只能用于单一治疗方案，大型动物数​​量，可能需要一个完整的研究。上述方法的一个共同特点是，它们确定平均血管通透性为所有的血管组织样本内。但是，它是公认的肺部微观和宏观血管表型不同9。因此，渗透性反应可能是异构的各种血管段以及9,10之间。因此，定量组织样本中的所有肺血管通透性均可能没有充分反映这种异质性。 在离体血液灌注肺标本，血管对肺表面可以通过一个堂堂正正的显微镜4,11,12可视化。这使得在单个容器的反应特征，因此，解决在应对13任异质性。此外，通过利用微血管的荧光成像，基于荧光测定法可并入。进一步，左心房微导管可被用于运载剂和荧光探针进入血管11,14。微导管限制了传递到小肺部区域，从而前冒充只有区域内的血管的灌注剂和荧光基团。这允许将用于单独的实验相同的肺中的多个小区域，导致所需的研究动物的整体减少。 即时成像能够在血管和微血管单隔离肺准备的荧光血管外动态变化的捕获。因此，对于像场中的每个微血管，输液荧光和冲刷的过程中发生变化的荧光可以被记录，并量化离线14。使用的最大和残余血管荧光值，摄像视场中的每个微血管通透性指数可以被确定。以确定响应于炎性或损害性药物的渗透性的变化，所需的剂可以首先给药，然后通透性指数来确定。此外，该图像字段可以设置为通过注入肺区域内微导管，从而使之在选择所需的血管网的高度灵活性。因此，孤立的血液灌注肺的准备以配合实时成像提供了一个有吸引力的实验模型来量化通透性单肺微血管。

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tygon Tubing</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:297px;">#18</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pressure Transducer</td>
			<td style="width:108px;">Data Sciences International</td>
			<td style="width:119px;">P23XL</td>
			<td>Need quantity 3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Butterfly Needle</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td align="right" style="width:119px;">450081</td>
			<td>21G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peristaltic pump</td>
			<td style="width:108px;">Cole Parmer</td>
			<td style="width:119px;">Masterflex L/S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-90 tubing</td>
			<td style="width:108px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">427421</td>
			<td>30 cm needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-10 tubing</td>
			<td style="width:108px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">427401</td>
			<td>40 cm needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe Pump</td>
			<td style="width:108px;">Braintree Scientific</td>
			<td style="width:119px;">BS8000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">O-ring</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>Custom made with a 20 mm diamter hole&#160; and a handle&#160; to secure o-ring to holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Upright fluorescence microscope</td>
			<td style="width:108px;">Olympus America</td>
			<td style="width:119px;">BX61WI</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Image Acquisition Software</td>
			<td style="width:108px;">Molecular Devices</td>
			<td style="width:119px;">Metamorph</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC Dextran 20KD</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>0.5 mg/ml (A dextran of different molecular size can be selected, if trial experiments indicate its suitability based on the calculated permeability index values)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lipopolysaccharide</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>Serotype 0111:B4</td>
		</tr>
	</tbody>
</table>

quantifying single microvessel permeability in isolated blood-perfused rat lung preparation

分离的血液灌注的肺制剂被广泛用于可视化和单微血管定义信令。通过实时成像耦合此制备中，以确定在单个肺微血管通透性的改变成为可能。在这里，我们描述的步骤来隔离大鼠肺组织，并与自体血灌注他们。然后，我们概述的步骤注入通过微导管荧光团或代理人到一个小细胞肺癌区域。使用上述这些程序，我们确定通透性增加大鼠肺微血管针对细菌脂多糖注射。数据显示，脂多糖通过这两个小静脉和毛细血管微血管段增加液体泄漏。因此，该方法使得有可能以比较的血管段中的渗透性的反应，因此，限定在响应中的任何不均匀性。而常用的方法来定义肺通透性要求肺组织样品，进行后处理的使用实时成像消除这一要求作为显而易见的，从目前的方法。因此，隔离肺制剂结合实时成像提供了一些优于传统方法来确定肺微血管的通透性，又是一个简单的方法来制定和实施。

分离的血液灌注肺制剂连接到灌注管道和相关设备示于图2。出于演示的目的，我们使用了一个只SD大鼠，虽然本文所描述的程序，可以用任何鼠种使用。通过左心房微导管输注到达肺的一小区域。该注入区域可通过输注引起的变色（ 图3）来识别。作为定位于实时成像的肺制剂示于图4。 <img alt="图2" fo:content-wid...

Watch this Scientific Journal Video about Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation at JoVE.com

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

孤立的血液灌注肺的准备，使可视化微血管网络上的肺表面是可行的。在这里，我们描述的方法用实时荧光成像量化单一微​​血管隔离肺的通透性。

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 ...

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JoVE Journal

Biology

9.4K 次观看.  The University of Tennessee Health Science Center. 孤立的血液灌注肺的准备，使可视化微血管网络上的肺表面是可行的。在这里，我们描述的方法用实时荧光成像量化单一微​​血管隔离肺的通透性。 

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

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

标准化的制备小鼠肺组织单细胞悬浮液使用gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

科研

生物学

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

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

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 ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

“体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞：隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

单通道分析和钙成像在新鲜分离的肾小球足细胞

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146的分离<sup&gt; +</sup&gt;从大鼠肺组织驻地肺间质基质细胞

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

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 ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

一种用于快速鉴定根系分泌物中根细菌化学引诱剂的改进趋化性测定

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...