KO和AC支持赠款和美国心脏协会的奖学金。 SIB和RB是支持由美国国立卫生研究院的资助。

<a href="https://www.jove.com/files/ftp_upload/1435/SOHA_overview.pdf">点击这里查看半自动化的心跳分析（所下）程序的概述</a> 。 半自动光学心跳分析 1。预处理 - 这一步提供舒张压和收缩压的心直径的信息。 心脏边缘识别​​和标记在最大的舒张和收缩。光学录音，可先进的速度缓慢，甚至在时间允许的最大收缩和放松的心发生的帧精确识别帧。一双识别舒张边缘和一对确定收缩压边缘的商标之一。商标也可一式两份，在不同的水平位置，沿心脏。 在此步骤中获得的心脏直径也用于计算一个额外的测量提供了一个心脏的收缩力的估计。这项措施是％，短轴缩短率（％FS），它代表了心脏边缘，朝对方移动过程中收缩程度。 ％FS用来计算公式是： （（（舒张末期内径 - 收缩直径）/舒张末期内径）× 100） 注 - 在此设置确定该点仅用于直径和缩短分数测量，不使用移动检测。 例 ：（。Cammarato，等2008）在果蝇中，肌球蛋白重链的传感器域点突变，一个地区的已知来调节电机的酶的活性，结果在心中，是扩张或限制取决于是否他们抑制或增强肌球蛋白的ATP酶活性的活动时。这些类型的测量不能完整的苍蝇，因为它是通常没有可能沿的心管通过的角质层和脂肪的整个长度的心边，（比较野生型，D45和Mhc5“中号的模式中号的模式图“下面），通常环绕的心。测量可视化与其他普遍采用的方法在完整的苍蝇的核心时，通常仅限于心脏的最前部分，锥形腔地区。它也不可能获得这个固定的组织中的动态测量。 2。移动检测这一步是自动完成的程序和使用两种不同的算法来分析每部电影的运动。简单地说，第一种算法，“平均帧黑暗”算法使用一帧一帧的方法。该算法平均为整个电影框架的黑暗强度，规范化的值0和1之间的时间间隔，随着时间的推移电影的每一帧图正常化平均。第二种方法，“像素”的算法，分析了在黑暗中的所有单个像素从一帧到下一变化。像素，表现出强度超过预设的阈值的变化概括为每个帧，并随着时间的推移电影的每一帧绘制。 3。检查间隔从两个运动检测算法的输出是“平均黑暗”在上面的输出窗口中的“像素”窗口显示的输出显示在此模块。为了确保检测的时间间隔与运动同意，在实际的电影，我们显示边缘跟踪或M模式，直接在下面分析该算法的输出窗口的帧。 M型是由数字切割片，通过心脏的一个像素宽在电影的每一帧，并调整水平提供的心随着时间的推移，边缘运动的快照。窗口中的“像素”所确定的舒张间隔上方促进该时间间隔的帧的数量与水平的绿线表示。收缩期间隔时间被确定为两个连续diastoles之间的时间间隔。收缩的开始，是一个垂直的蓝线表示在M模式，到底是由一个红色的垂直线表示。有了这个显示，它是比较容易看到算法的输出，如果同意与实际的心脏运动。 虽然运动检测是自动执行的输出必须检查的准确性。有几个原因之一是，往往有一个潜在的，在不相关的心脏运动的光线水平慢波波动。这通常可以使用一个内置的高通滤波器消除。第二个原因与灵敏度的“像素”像素“的方针。从这种算法的输出通常是两个运动的痕迹，收缩运动和放松运动之一。这两个运动之间的时间长度在年纪较大的苍蝇和一些突变体<html>变得比较长，程序试图解释为两个单独的节拍的两个动作。可以纠正这种情况，提出的方案，比较像素的输出像素算法的平均帧黑暗算法，通过点击“使用黑暗”框中。 范例（见视频）：在5至7周龄苍蝇收缩间隔通常是更长的时间比在年轻的苍蝇。最初发现这些长的时间间隔短的舒张分离的两个独立的收缩。可以纠正这种情况，选择“使用黑暗”复选框。在这种情况下，程序将比较舒张间隔的平均帧黑暗输出像素与像素算法确定。如果平均帧黑暗输出具有积极的价值，那么任何舒张间隔像素像素算法确定将被忽略，并且将正确地指定的时间间隔。整个影片可以在10秒段扫描，并接受（点击“数据行”）或拒绝（点击“丢弃数据”）。 HI和LO通过过滤器的灵敏度可手动调整，以帮助更准确地定义收缩的间隔时，用人黑暗的算法。 4。产量统计一旦已被接受为一个给定的影片的输出程序将使用这些信息自动计算参数的数量。这些措施包括收缩压和舒张压的时间间隔，心脏病期和心率，方向和速度的收缩波，心脏直径，和％FS。此外，该计划已量化arrhythmicity反映非均匀的收缩和舒张周期（见芬克等，2009）的多种方法。统计输出是单独为每部电影和整个数据形成一个逗号分隔值（CSV）文件，如Microsoft Excel电子表格程序，可在打开的设置。 表1 - <img src="/files/ftp_upload/1435/1435table1.jpg" alt="表1" /> 半自动化光学心脏分析程序生成的输出的一个例子。 5。更新直方图; 这部分的程序对心脏间隔的数据显示直方图格式的数据集。由于心脏率是可变的的，我们直方图的输出数据正常化。然而，因为间隔的分布是不围绕着对称的意思是我们用我们正常化的时间间隔中位数值。 示例（见视频）：KCNQ钾离子通道突变的果蝇表现出极不规则的舒张压和收缩压的时间间隔。通常情况下收缩间隔更长的时间，超过年龄相匹配的野生型果蝇的不规则。可以清楚地看出这显示直方图格式的数据（也看到Ocorr等。2007年）。 6。 M模式此模块产生的M -模式，可从任何位置沿动画帧的心脏。还可以指定长度的M -模式。 M -模式是很有用的显示质心在电影做。 范例（见视频）：3天的斑马鱼幼体显示前心室的心，是用来产生一个M -模式的电影。将电子从每部电影帧切除像素的垂直切片显示一条红线。 M型图 ：从果蝇和斑马鱼的M -模式的例子。与更快的酶，从而导致心脏限制动力学和偶尔传导阻​​滞的分子马达的预处理步骤结果讨论了肌球蛋白基因突变。这两个参数可使用M -模式显示定性。 <img src="/files/ftp_upload/1435/1435fig1.jpg" alt="M -模式的例子" /> 在野生型和突变的果蝇的心脏在同一部分的M -模式清楚地显示在MHC 5突变体和扩张的直径和D45突变（对酶动力学低迷的肌球蛋白）心律失常收缩周期限制的表型（修改从Cammarato等人，2008年）。 M模式从斑马鱼幼虫前心室的心脏（同样的心的电影，在演示中，芬克等人2009所示）。 7。红点电影这个模块会自动产生一个放缓（1:4），20的分析显示，在红色改变标识的所有像素的影片的第二个版本。此功能主要是用于说明目的。 电影1 -一个“红点电影”显示，有红色的程序算法的黑暗强度的变化来确定的像素。影片从原来的4倍的速度放缓。 w.jove.com/files/ftp_upload/1435/redmove.avi“&gt;点击这里下载电影1。 8。额外的应用程序我们已经开始了这一分析系统，适用于小的心，这是经常使用传统方法难以分析其他型号的高速动画。我们已经成功地应用这一分析的斑马鱼的幼体心中胚胎小鼠心脏（芬克等人，2009年）。 电影2 -小鼠心脏电影加数字数据输出的检查间隔。 <a href="https://www.jove.com/files/ftp_upload/1435/Mouse%20heart%20clip%201.avi">点击这里下载电影2。</a> <img src="/files/ftp_upload/1435/1435fig2.jpg" alt="检查小鼠心脏间隔" />

<ol>
	<li>Cammarato, A., Dambacher, C. M., Reedy, M. C., Knowles, A. F., Kronert, W. A., Bodmer, R., Ocorr, K., Bernstein, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+Transducer+mutations+differentially+affect+motor+function,+myofibril+structure,+and+the+performance+of+skeletal+and+cardiac+muscles.">Myosin Transducer mutations differentially affect motor function, myofibril structure, and the performance of skeletal and cardiac muscles.</a> Mol Biol Cell. 19 (2), 553-562 (2008).</li><li>Ocorr, K., Reeves, N., Wessells, R. J., Fink, M., Chen, H. -. S. V., Akasaka, T., Yasuda, S., Metzger, J., Giles, W., Posakony, J. W., Bodmer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCNQ+potassium+channel+mutations+cause+cardiac+arrhythmias+in+Drosophila+that+mimic+the+effects+of+aging.">KCNQ potassium channel mutations cause cardiac arrhythmias in Drosophila that mimic the effects of aging.</a> Proc Natl Acad Sci U S A. 104, 3943-3948 (2007).</li><li>Fink, M., Callol-Massot, C., Chu, A., Ruiz-Lozano, P., Izpisua Belmonte, J. C., Giles, W., Bodmer, R., Ocorr, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+detection+and+quantification+of+heartbeat+parameters+in+Drosophila,+zebrafish+and+embryonic+mouse+hearts.">A new method for the detection and quantification of heartbeat parameters in Drosophila, zebrafish and embryonic mouse hearts.</a> Biotechniques. 46, 101-113 (2009).</li></ol>

The authors have nothing to disclose.

果蝇模型已被证明是一个强大的遗传工具，已经被用来解决各种科学问题，从胚胎发育，学习和记忆不等。最近这种多功能的模式生物用于研究心脏功能的基因。一些试图量化在成年果蝇的心脏生理依赖于完整的苍蝇经腹角质层的意见。这些方法大多依靠目视观察，或通过腹部量化一个参数，心率传输光的强度变化的录音。虽然这是一个有用的参数，它是有限的，它告诉我们对心脏功能。我们的?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>MatLab software</td>
<td>&nbsp;</td>
<td>Math Works, Inc</td>
<td>&nbsp;</td>
<td>Required environment for the analysis software.</td>
</tr>
<tr>
<td>EM-CCD digital camera</td>
<td>&nbsp;</td>
<td>Hamamatsu Corp.</td>
<td>9100 or 9300</td>
<td>Other high speed digital cameras will also work.</td>
</tr>
<tr>
<td>HC image data capture software</td>
<td>&nbsp;</td>
<td>Hamamatsu Corp.</td>
<td>&nbsp;</td>
<td>Other image capture software that produces movies in avi format will also work.</td>
</tr>
<tr>
<td>Light Microscope with 10x objective </td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>A dipping lens that eliminates the air water interface greatly improves resolution</td>
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semi-automated optical heartbeat analysis of small hearts

我们已经开发出一种用于分析高速光学录音方法<em&gt;果蝇</em&gt;，斑马鱼和小鼠胚胎心（芬克等，2009）。我们的半自动光学心跳分析（所下）使用一种新的运动检测算法，该算法能够检测与个别的收缩和放松活动相关的心脏运动。该方案提供了有关生理的读数，包括收缩压和舒张压的时间间隔，心率，以及定性和定量的心跳arrhythmicity主机。该方案还计算出在心脏舒张和收缩缩短分数和小数的面积变化计算的直径测量。输出为数字文件，与大多数电子表格程序兼容。每一次心跳的测量是在提高统计输出功率纪录。我们证明每个用户输入所需的步骤，并显示我们的方法的应用在所有这三个基因听话的心脏模型的心功能分析。

Watch this Scientific Journal Video about Semi-automated Optical Heartbeat Analysis of Small Hearts at JoVE.com

Semi-automated Optical Heartbeat Analysis of Small Hearts

我们已经开发了一个半自动化光学检测信号分析方法（所下分析高速光学录音）<em&gt;果蝇</em&gt;，斑马鱼和小鼠胚胎心中。我们证明我们的方法在果蝇和小鼠胚胎心脏的心脏功能分析中的应用。

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

semi-automated-optical-heartbeat-analysis-of-small-hearts

Research

JoVE Journal

Biology

12.1K 次观看.  The Sanford Burnham Institute for Medical Research. 我们已经开发了一个半自动化光学检测信号分析方法（所下分析高速光学录音）果蝇，斑马鱼和小鼠胚胎心中。我们证明我们的方法在果蝇和小鼠胚胎心脏的心脏功能分析中的应用。

视频: Semi-automated Optical Heartbeat Analysis of Small Hearts

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

适当的照顾和清洁的显微镜

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

科研

生物学

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

光镜下的主要组成部分

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

相衬，微分干涉对比（DIC）显微镜

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

光学测绘的Langendorff灌注大鼠心脏

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the primary photosynthetic organ, and their size and shape vary developmentally and environmentally within a plant. For these reasons studies on leaf morphology require measurement of multiple parameters from numerous leaves, which is best done by semi-automated phenomics tools5,6. Canopy shade is an important environmental cue that affects plant architecture and life history; the suite of responses is collectively called the shade avoidance syndrome (SAS)7. Among SAS responses, shade induced leaf petiole elongation and changes in blade area are particularly useful as indices8. To date, leaf shape programs (e.g. SHAPE9, LAMINA10, LeafAnalyzer11, LEAFPROCESSOR12) can measure leaf outlines and categorize leaf shapes, but can not output petiole length. Lack of large-scale measurement systems of leaf petioles has inhibited phenomics approaches to SAS research. In this paper, we describe a newly developed ImageJ plugin, called LeafJ, which can rapidly measure petiole length and leaf blade parameters of the model plant Arabidopsis thaliana. For the occasional leaf that required manual correction of the petiole/leaf blade boundary we used a touch-screen tablet. Further, leaf cell shape and leaf cell numbers are important determinants of leaf size13. Separate from LeafJ we also present a protocol for using a touch-screen tablet for measuring cell shape, area, and size. Our leaf trait measurement system is not limited to shade-avoidance research and will accelerate leaf phenotyping of many mutants and screening plants by leaf phenotyping.

Demonstration of key methods for high throughput leaf measurements. These methods can be used to accelerate leaf phenotyping when studying many plant mutants or otherwise screening plants by leaf phenotype.

LeafJ：ImageJ的插件半自动叶形状测量

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and &#945;-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

从固定心脏分离心肌细胞进行免疫细胞化学和倍体分析

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably ...

Quantitative Analysis of Viscoelastic Properties of Red Blood Cells Using Optical Tweezers and Defocusing Microscopy

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not only attributed to the normal variability of cells, but also to the differences in methods and models of cell response. Here, an integrated protocol using optical tweezers and defocusing microscopy is employed to obtain the rheological features of red blood cells in the frequency range of 1 Hz to 35 Hz. While optical tweezers are utilized to measure the erythrocyte-complex elastic constant, defocusing microscopy is able to obtain the cell height profile, volume, and its form factor a parameter that allows conversion of complex elastic constant into complex shear modulus. Moreover, applying a soft glassy rheology model, the scaling exponent for both moduli can be obtained. The developed methodology allows to explore the mechanical behavior of red blood cells, characterizing their viscoelastic parameters, obtained under well-defined experimental conditions, for several physiological and pathological conditions.

Here, an integrated protocol based on optical tweezers and defocusing microscopy is described to measure the rheological properties of cells. This protocol has wide applicability in studying the viscoelastic properties of erythrocytes under variable physio-pathological conditions.

使用光学镊子和散焦显微镜定量分析红细胞的粘弹性

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not ...

小鼠白色脂肪组织的半自动分离

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

作者聚焦：使用组织解离剂从小鼠白色脂肪组织中半自动分离基质血管组分  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...