本研究由中国国家重点研究开发项目 (2017YFC1700503)、全国基础研究计划 (973 计划)、中国国家自然科学基金 (81370223 号) 资助。81573957号), 以及北京协和医学院研究生创新研究基金会 (2016-1002-01-02)。

1. 试剂和溶液的制备
<ol><li>根据制造商的指示, 加入25毫升肌细胞生长培养基补充剂, 以500毫升的细胞生长培养基, 制备人心肌肌 (母国) 全培养基。在使用前贮存在4°c 和温暖到37°c。</li>
	<li>将 txl-特格尔超细粉溶于血清/葡萄糖 Dulbecco 修饰鹰的培养基 (DMEM) 中, 制备通心络 (txl-特格尔) 溶液, 并通过添加 txl-特格尔将µg 浓度调整为 400 DMEM/毫升 (详情见11)。在使用前贮存在4°c 和温暖到37°c。</li>
	<li>在黑暗中, 在15毫升离心管中加入25µL JC-1 溶液 (200x)、4毫升超纯水和1毫升的库存染色缓冲器 (5x), 准备5毫升的 JC-1 工作溶液。在使用前用吸管将混合物上下数次, 加热到37摄氏度。</li>
	<li>在50毫升离心管中加入24毫升的超纯水到6毫升的库存染色缓冲器 (5x), 制备30毫升工作染色缓冲器。使用此解决方案冰冷。</li>
</ol>2. 建立缺氧/复氧模型
<ol><li>板 1.0 x 105细胞每井与 HCM 完全媒介在6个井板材。生长达到约70% 的融合在细胞孵化器包含 5% CO2和95% 空气在37°c。</li>
	<li>用磷酸盐缓冲盐水 (PBS) 清洗母国措施。使他们接触不同的治疗方法 (400 µg/毫升 txl-特格尔或不) 在2毫升血清/葡萄糖无 DMEM 30 分钟缺氧前的细胞孵化器包含 5% CO2和95% 空气37°c。</li>
	<li>在密闭和缺氧的罐子 (2.5 升) 上孵化母国措施, 装有催化剂来清除游离氧, 并诱导18小时缺氧 (图 1A), 以及用于测量介质11、12中氧张力的厌氧指示器,13, 在一个细胞孵化器包含 5% CO2和95% 空气在37°c。 注: 催化剂在罐中清除自由氧需要大约1.5 小时。因此, 在罐子打开之前, 母国措施在罐子里保持19.5 小时。在缺氧条件下, 厌氧指示器的颜色由淡蓝色变化为常氧白色 (图 1B)。</li>
	<li>Reoxygenate 母国措施将6井板块移动到一个孵化器设置在37°c, 包含 5% CO2和95% 空气 2 h。</li>
</ol>3. 流式细胞术用母国措施的研制
<ol><li>将0.25% 胰蛋白酶-乙二胺四乙酸 (EDTA) 溶液和 PBS 加热至37摄氏度。</li>
	<li>从各井中抽出培养基, 用1毫升 PBS 冲洗细胞, 并在井壁上加0.5 毫升0.25% 胰蛋白酶。</li>
	<li>孵化37摄氏度, 直到所有的母国措施几乎分离 (约1分钟)。</li>
	<li>添加1毫升的 DMEM 完全培养基 (DMEM 培养基和10% 胎牛血清) 到井中中和胰蛋白酶。在室温下将细胞悬浮液转移到15毫升离心管和颗粒离心, 在 500 x g 处进行3分钟。</li>
	<li>小心地吸入上清液而不干扰颗粒。</li>
	<li>添加0.5 毫升的 HCM 完整培养基和0.5 毫升的 JC-1 工作溶液, 每管。并用重悬吹打。</li>
	<li>用铝箔覆盖整个管子, 以防止荧光指示器的漂白和在正常情况下 (在37摄氏度含 5% CO2和95% 空气中) 的情况下在20分钟内孵化母国措施。</li>
	<li>颗粒母国措施由离心在 500 x g 3 分钟在4°c。</li>
	<li>用2毫升冰冷 JC-1 染色缓冲器冲洗两次母国措施。</li>
	<li>在样品管 (12 毫米 x 75 毫米) 的最后一卷300µL 的冰冷染色缓冲器中并用重悬母国措施, 并在30分钟内使用细胞进行分析。 注: 使用羰基氰化物 m-氯苯基腙 (CCCP), 一种有效的呼吸电子转运链抑制剂, 治疗1小时的母国措施, 浓度为20µM, 为阳性对照。</li>
</ol>4. 流式细胞仪设置
<ol><li>启动流式细胞仪, 并确保软件连接到它。</li>
	<li>执行射流技术启动, 启动流并设置 breakoff。</li>
	<li>在流式细胞术软件中打开两个点绘制窗口。<ol>
<li>选择在第一个点图中 Y 轴上的 X 轴和侧散射光 (SSC) 上的正散射光 (FSC), 分别表示单元格的大小和粒度。</li>
		<li>使用第二点图中的对数刻度为 X 轴选择 PE (575 英寸 26 nm) 检测通道, 用于测量 FITC 中的 JC-1 染料的荧光强度。</li>
	</ol>
</li>
</ol>5. 流式细胞仪测量和数据分析
<ol><li>单击 "卸载", 释放管道支撑臂并定位空白(未用 JC-1 染色的母国措施)。单击 "加载", 将管道支撑臂返回到其工作位置。</li>
	<li>单击 "记录" 从空取样管中的悬浮中收集粒子, 然后在第一个点图中对单元格填充 (P1) 进行进一步分析 (图 3)。</li>
	<li>收集其他取样管的母国措施, 通过调整荧光通道的电压来优化测量, 以确保单元格点位于第二点图的中心区域。</li>
	<li>通过将红荧光强度的比值除以绿色荧光强度, 显示各样本管的统计值, 并计算出每个样品的基质金属蛋白酶。</li>
</ol>

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作者没有什么可透露的。

在这里, 我们提出一个协议, 使用 JC-1 染料来评估细胞的基质干细胞暴露后, 通过 JC-1 检测发现, 细胞的基质干细胞是独立的因素, 如线粒体大小, 形状和密度可能影响单组分荧光信号14。因此, JC-1 化验结果比较可靠。同时, 对 JC-1 的检测具有很方便、省时的性能。这种检测对材料和试剂的要求很低, 一般情况下, 从细胞分离到基质金属蛋白酶的测量, 其染色需要少于1.5 小时。
<p cl...

冠心病是全世界死亡的主要原因。ST 段抬高心肌梗死患者减少缺血性损伤和限制梗死大小的选择是通过经皮冠状动脉介入治疗 (PCI) 的及时有效的心肌再灌注1, 2。然而, 再灌注导致额外的损伤, 可能占到最后梗塞大小的30% 的3。普遍认为线粒体通透性过渡孔隙 (mPTP) 不仅是缺血/再灌注过程中线粒体损伤和细胞死亡的中枢, 而且也是心肌梗死信号4的收敛目标。,5. 随着 mPTP 的开放将导致内线粒体膜电位的退极化 (基质金属蛋白酶)4, 我们检测到 mPTP 开放使用 5,5′, 6,6′四氯化碳 1,1′, 3,3′-四乙基 imidacarbocyanineiodide (JC-1) 化验。
JC-1 法是一种定性和定量的 cytofluorimetric 方法, 通过对单个线粒体6级的基质细胞进行分析, 进一步验证了它的有效性。JC-1 作为一种聚集形式存在, 在线粒体基质中产生红色到橙色的辐射 (590 17.5 nm);随着基质的丧失, JC-1 被转化为单体形式, 产生绿色荧光, 发射量为 530-15 nm。因此, 红/绿荧光强度比的降低可以表明在缺血/再灌注 (I/R) 条件下基质金属蛋白酶的减少。
除了 JC-1, 还研究了膜渗透亲脂阳离子, 如罗丹明123和 3,3′-dihexyloxadicarbocyanine 碘 [DiOC6 (3)]。然而, 与这两种探头相比, JC-1 更能可靠地分析基质金属蛋白酶。罗丹明123具有相对较差的灵敏度 (特别是在淬火方式7,8) 和较差的特异性。在罗丹明123的转变有时是如此之小, 研究人员或设备很难观察/检测。此外, 在单个细胞中, 罗丹明123有不同的线粒体结合点, 因此它可能有不同的荧光排放量9。DiOC6 (3) 不推荐用于检测基质金属蛋白酶, 因为它对等离子体膜10的退极化反应灵敏。
因此, 在这里, 我们使用 JC-1 的方法来评估在接触到缺氧/复氧或没有保护剂的情况下母国措施的基质。

<table border="0" cellpadding="0" cellspacing="0" width="970">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">Mitochondrial membrane potential assay kit with JC-1</td>
			<td style="width:307px;">Beyodtime, China</td>
			<td style="width:80px;">C2006</td>
			<td style="width:291px;">In the kit there are JC-1 stock solution (200&#215;), stock staining buffer (5&#215;) and CCCP(10mM)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tongxinluo ultrafine powder</td>
			<td>Shijiazhuang Yiling Pharmaceutical Co., China</td>
			<td>071201</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">Annexin V-FITC/PI Kit</td>
			<td>Becton-Dickinson, USA</td>
			<td>556547</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM</td>
			<td>Life Technologies, Grand Island Biological Company, USA</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Human cardiac myocyte</td>
			<td>Promocell, Germany</td>
			<td>C-12810</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">Myocyte Growth Medium 
			(SupplementMix)</td>
			<td>Promocell, Germany</td>
			<td>C-39275</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Myocyte Growth Medium (Ready-to-use)</td>
			<td>Promocell, Germany</td>
			<td>C-22070</td>
			<td>used with Myocyte Growth Medium SupplementMix</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GENbox</td>
			<td>BioM&#233;rieux, Marcy l&#8217;Etoile, France</td>
			<td>96127</td>
			<td>2.5L</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Catalyst (AnaeroPack)</td>
			<td>MITSUBISHI GAS CHEMICAL&#160;COMPANY, INC.&#160;, Japan</td>
			<td>&#160;C-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anaerobic indicator</td>
			<td>BioM&#233;rieux, Marcy l&#8217;Etoile, France</td>
			<td>96118</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow cytometer</td>
			<td>Becton-Dickinson, USA</td>
			<td>FACSAria 2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BD FACSDiva Software</td>
			<td>Becton-Dickinson, USA</td>
			<td></td>
			<td>Version8.0.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sample tube</td>
			<td>Corning science, USA</td>
			<td>352054</td>
			<td>12*75mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS</td>
			<td>Hyclone, USA</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
	</tbody>
</table>

a flow cytometry-based assay for measuring mitochondrial membrane potential in cardiac myocytes after hypoxia/reoxygenation

及时有效地再灌注闭塞性冠状动脉是降低 st 段抬高心肌梗死患者心肌梗死大小的最佳策略。然而, 再灌注本身可能导致进一步的心肌细胞死亡, 这一现象被称为再灌注损伤。线粒体通透性过渡孔隙 (mPTP) 的开放, 随着线粒体膜电位 (基质) 的减少, 或线粒体的退极化, 被普遍认为是再灌注损伤的最后一步, 负责线粒体和心肌细胞死亡。JC-1 是一种脂溶性阳离子染料, 在线粒体中积累取决于基质的价值。基质金属蛋白酶越高, 线粒体中的 JC-1 积累越多。线粒体中 JC-1 含量的增加可以通过荧光发射从绿色 (~ 530 nm) 到红色 (~ 590 nm) 来反映出来。因此, 降低红/绿荧光强度比可以表明线粒体的退极化。在这里, 我们利用 JC-1 测量基质金属蛋白酶, 或在缺氧/复氧后的人心肌细胞中 mPTP 的开放, 通过流式细胞术检测。

在进行 JC-1 试验以评估基质金属蛋白酶的变化之前, 强烈建议进行实验以确认研究人员成功确定的条件。如流式细胞术结果 (图 2) 所示, 与正常组相比, 缺氧/复氧 (H/r) 显著诱导了母国措施 (蛋白 V +/PI±) 的凋亡, 表明我们建立了一个基于细胞的 I/R (45.00 2.13% vs) 模型。11.50, 0.18% 在正常组, p < 0.05)。通心络是一种具有心肌作用的中药, 在 h/r 组 ( p ...

Watch this Scientific Journal Video about A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation at JoVE.com

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

在这里, 我们提出一个协议, 使用 JC-1 染料评估细胞线粒体膜电位后, 暴露在缺氧/复氧与或没有保护剂。

流式细胞仪测定缺氧/复氧后心肌细胞线粒体膜电位的实验研究

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

a-flow-cytometry-based-assay-for-measuring-mitochondrial-membrane

Research

JoVE Journal

Medicine

14.1K 次观看.  Chinese Academy of Medical Sciences and Peking Union Medical College. 在这里, 我们提出一个协议, 使用 JC-1 染料评估细胞线粒体膜电位后, 暴露在缺氧/复氧与或没有保护剂。

视频: A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Children

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory rate, salivation, perspiration and renal function. The ANS has two main branches: the sympathetic nervous system, preparing the human body for action in times of danger and stress, and the parasympathetic nervous system, which regulates the resting state of the body.
ANS activity can be measured invasively, for instance by radiotracer techniques or microelectrode recording from superficial nerves, or it can be measured non-invasively by using changes in an organ's response as a proxy for changes in ANS activity, for instance of the sweat glands or the heart. Invasive measurements have the highest validity but are very poorly feasible in large scale samples where non-invasive measures are the preferred approach. Autonomic effects on the heart can be reliably quantified by the recording of the electrocardiogram (ECG) in combination with the impedance cardiogram (ICG), which reflects the changes in thorax impedance in response to respiration and the ejection of blood from the ventricle into the aorta. From the respiration and ECG signals, respiratory sinus arrhythmia can be extracted as a measure of cardiac parasympathetic control. From the ECG and the left ventricular ejection signals, the preejection period can be extracted as a measure of cardiac sympathetic control. ECG and ICG recording is mostly done in laboratory settings. However, having the subjects report to a laboratory greatly reduces ecological validity, is not always doable in large scale epidemiological studies, and can be intimidating for young children. An ambulatory device for ECG and ICG simultaneously resolves these three problems.
Here, we present a study design for a minimally invasive and rapid assessment of cardiac autonomic control in children, using a validated ambulatory device 1-5, the VU University Ambulatory Monitoring System (VU-AMS, Amsterdam, the Netherlands,<a href="http://www.vu-ams.nl" target="_blank"> www.vu-ams.nl</a>).

Measuring Cardiac Autonomic Nervous System ANS Activity in Children

Measurement of autonomic nervous system activity usually confines the researcher and participant to the laboratory, which may provide an intimidating environment to children. The VU University Ambulatory Monitoring System (VU-AMS) device can record cardiac autonomic control in any setting. The VU-AMS proved very amenable to testing in children.

测量心脏自主神经系统（ANS）儿童活动

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory ...

科研

医学

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

单元位置的Imaging-和流式细胞仪为基础的分析和3D球体黑色素瘤细胞周期

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

从健康志愿者和它们的迁徙可能通过血清中的影响心脏手术后的内皮祖细胞的分离

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) function are monitored by the change in voltage across the nasal epithelium after the superfusion of solutions that modify ion channel activity. This is enabled by the measurement of the potential difference between the subcutaneous compartment and the airway epithelium in the nostril, utilizing a catheter in contact with the inferior nasal turbinate.
The test allows the measurement of the stable baseline voltage and the successive net voltage changes after perfusion of 100 &#181;M amiloride, an inhibitor of Na+ reabsorption in Ringer&#39;s solution; a chloride-free solution containing amiloride to drive chloride secretion and 10 &#181;M isoproterenol in a chloride-free solution with amiloride to stimulate the cyclic adenosine monophosphate (cAMP)-dependent chloride conductance related to CFTR.
This technique has the advantage of demonstrating the electrophysiological properties of two key components establishing the hydration of the airway surface liquid of the respiratory epithelium, ENaC, and CFTR. Therefore, it is a useful research tool for phase 2 and proof of concept trials of agents that target CFTR and ENaC activity for the treatment of cystic fibrosis (CF) lung disease. It is also a key follow-up procedure to establish CFTR dysfunction when genetic testing and sweat testing are equivocal. Unlike sweat chloride, the test is relatively more time consuming and costly. It also requires operator training and expertise to conduct the test effectively. Inter- and intra-subject variability has been reported in this technique especially in young or uncooperative subjects. To assist with this concern, interpretation has been improved through a recently validated algorithm.

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure.

鼻膜 Transepithelial 电位差的标准化测量 (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) ...

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

几内亚猪模型心肌损伤后 hiPSC-derived 衍生心肌贴片的植入

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

从成年小鼠分离心膜肌细胞

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

流循环模型中血液接触植入物的血液相容性测试模拟人类血流

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

人类心脏肌细胞模型中体温过低预科后心肌保护体外评估

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...