这项工作是由威康信托基金会，英国医学研究理事会，英国皇家学会科学城，伯明翰和不孕不育研究信托基金资助。我们要感谢山研究的专家的协助下，在建设和整合成像钻机。

从健康生育男性的精子与正常的精液分析，通常准备如下成像。 <ol><li>精液样本存储在37 ° C，不超过30分钟。细胞是孤立的精浆游泳进入补充Earle的平衡盐的解决方案（sEBSS起来; mM的：1.8氯化钙2 .2 H 2 Ø，5.37氯化钾，0.81硫酸镁4 .7 H，26.2碳酸氢钠3，1.0的NaH 2 PO 4。 · 2H 2 O，116.4氯化钠，55.6 D -葡萄糖，2.73娜丙酮酸，乳酸41.8娜）辅以0.3％的木炭de-lipidated/fatty酸免费分数V牛血清白蛋白（BSA的质量是成功的精子获能的关键）。 1毫升sEBBS吸管到每一个系列的5毫升管，轻轻地用0.3毫升精液underlayered。经过潜伏期为1小时（37 ° C，6％的CO 2）0.7毫升，轻轻地从每管和汇集。 10μL精子悬液稀释90μL的1％（V / V）福尔马林固定的细胞，那么精子计数在纽鲍尔室。然后调整细胞密度在暂停（含sEBSS）至600万细胞/ ml。 </li><li>样本，然后分为200μL分装松散皑皑管和孵育（37℃; 6％的CO 2），允许在5-6 h获能。 </li><li>盖玻片（22x50毫米）先前已与D -聚赖氨酸处理。 10μLD -聚赖氨酸溶液（10％W / V）是适用于盖玻片中心的小水珠。聚- D -赖氨酸然后让其自然风干。这可以在加热阶段，并应完全干燥。盖玻片附有一个封闭的，专用的，pe​​rfusable，聚碳酸酯成像室（尺寸35毫米× 20毫米× 5毫米;容量≈180μL）。华纳RC20室类似D -聚赖氨酸的真空润滑脂涂层盖玻片形式当细胞被认为在一个倒置显微镜和一个直径为12毫米的圆形盖玻片上表面形式的会议厅，使光的传输室的基础。 </li><li>俄勒冈州绿BAPTA1 - AM（OGB）或钙绿1 - AM是用于标签的细胞。 OGB准备在DMSO溶解。聚醚酸F127（清洁剂）是包括在二甲基亚砜，以防止“聚集”的染料。这可以准备的实验室（100毫克0.5毫升DMSO聚醚），使用前，或可以购买预先准备。 20μL添加到OGB 50微克（2.5毫克/毫升）等分。小瓶然后可以存储以备后用冷冻和解​​冻。 </li><li>精子获能后（5-6小时，在sEBBS 37 ° C，6％的CO 2）管成像和1.2μLOGB解决方案中选择添加，给予终浓度为10μm 。管然后再孵育40分钟后，将细胞悬液轻轻地注入到一个P1000（蓝色）枪头成像室流入端口。会议厅内放置在孵化器，D -聚赖氨酸涂层的盖玻片一侧向下，20分钟。细胞往往游泳沿室的表面，将坚持以D -聚赖氨酸涂层的面积。 </li><li>现在会议厅内安装在显微镜上，灌注设备，并连接到灌注约0.5毫升/分钟。滚子泵饲料生理盐水出境口岸进入会议厅和溢出是一个陷阱吸泵中删除。离开的准备工作至少10分钟，在黑暗中，而松散的细胞和细胞外染料灌注室中删除。温度稳定性是非常重要的的，因为小的波动， 不仅可能影响细胞的 Ca 2 +稳态，但也可能会影响K的D的染料。实验，一般在25℃，维持在这个级别的黑暗的房间温度达到。我们发现，室温保持在所要求的水平，提供比使用一个温度控制的阶段或盐水流入温控加热器更高的温度和重点稳定性。 </li><li>细胞被认为在选择目标和成像领域的相衬（40X或60X油浸）。 D -聚赖氨酸涂布面积的进气口附近的部分是首选，生理盐水流动是层流和一致。同样重要的是使用面积是适当的细胞密度（密度过度影响质量和数据的完整性，通过相邻细胞的信号皮卡）和细胞充分附着，但鞭毛的活动是明显的。相衬图像将被保存。该显微镜，然后切换时间缩短为获得清晰的荧光图像（通常是50-200毫秒）所需的最低荧光模式（激发480 nm处;排放量为540 nm）和曝光。 </li><li>实验开始激活的时间序列图像采集软件（IQ）。通常，图像采集0.1 Hz和光照是限制图像采集期间只使用一个softwaRE控制快门。可以使用更大的图像采集率，但必须褪色的问题，由于光损伤的细胞（见讨论）代文物津贴。智商（和其他大多数采集软件平台）将允许在实验过程中实时荧光图形。 </li><li>经过3-5分钟，持续时间盐碱成分操纵（如的[Ca 2 +] O）和应用药物控制期间是实现替代生理盐水灌注头。此外每个刺激的时间，使成像室的到来的时间可以计算，这里描述（在0.1赫兹采样）的反应缓慢的评估提供足够的精度。加入直接刺激盐水流量通过第二流入华纳RC20厅提供的端口，可以实现更高的精度更迅速的反应。 </li><li>图像分析脱机状态下使用的智商。利益（投资回报）​​的地区是一轮每个单元格（或单元的一部分），并绘制细胞区是由软件选择（自动背景减法。图像序列，然后检查，以去除细胞死亡或接受精子顶体反应（这似乎在实验过程中，那些表现出足够的运动时间序列分析时造成的文物的细胞质染料亏损）作为荧光大和快速增加。 </li><li>时间的荧光强度系列，然后获得每个投资回报率和离线在Excel中，这些数据进行分析。最初荧光标准化控制期间取得的平均值，用公式R = [（FF 休息 ）/ 休息 ] × 100％，其中R％荧光的变化，控制期间相比，F是荧光强度在时间t和F 的其余部分是控制期间获得至少30的F测定的平均值。 </li></ol>代表性的成果图1显示的图像与3微米的孕激素刺激细胞实验系列。在灰度显示上排和电影1的荧光图像和图所示相同的法师（电影2）伪（查找表&#39;16_colors在图像J），其中“酷”的颜色表明荧光强度低， “温暖”的颜色表示荧光强度高。细胞可视为在伪实验过程中，使用查找表的智商，但使用灰度一般比较翔实的评估。细胞是否标记。前两个图像采集0秒和100秒后开始录音。荧光应稳定，而且往往在后顶体面积和精子颈部。孕酮进入约175小号成像室和3和第 4 次的图像（180 s和220章第），显示了快速增长的[Ca 2 +] i的（荧光），原产于后头部和颈部区域的细胞。随后的图像是290，360，740，990和1440小号，显示初始衰减的[Ca 2 +] i的瞬态和一所中学的持续海拔发展。图2显示了一个电子表格分析，原始荧光值转换为每个归荧光的投资回报率（％相比，这一控制时期的变化，荧光），图3显示了时间标准化荧光图3显示“典型”的反应，3微米的孕激素的细胞。 <img src="/files/ftp_upload/1996/1996fig1.jpg" alt="图1" /> 图1。示例数据从一个细胞与孕激素的刺激。一系列灰度（上排）和伪（下排）的荧光图像显示。时间点0，180，220，290，360和990s。 3微米的孕激素进入成像室，在175号投资回报是第一个面板中显示。 <img src="/files/ftp_upload/1996/1996fig2.jpg" alt="图2" /> 图2：在Excel中的单细胞荧光数据分析。在黑色的数字荧光数据的时间序列，垂直排列。红色数字（下）都用在文本中的方程获得的规范化的数据。图表显示归为64细胞的荧光在这个实验中，刺激后，显示在荧光瞬态一个缓慢的上升和一系列小的振荡上升的情节。 <img src="/files/ftp_upload/1996/1996fig3.jpg" alt="图3" /> 图3。时间的痕迹，3个单个细胞的荧光表达方面控制值增减％... 3微米孕酮是由箭头标记的时间。虚线显示平均控制荧光。

<ol>
	<li>Haughland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Fluorescent Probes and Research Products. , (2002).</li><li>Nishigaki, T., Wood, C. D., Shiba, K., Baba, S. A., Darszon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroboscopic+illumination+using+light-emitting+diodes+reduces+phototoxicity+in+fluorescence+cell+imaging.">Stroboscopic illumination using light-emitting diodes reduces phototoxicity in fluorescence cell imaging.</a> Biotechniques. 41, 191-197 (2006).</li></ol>

当成功地进行了，这种技术允许自发和诱发的Ca 2 +信号在人类精子的分布和动力学的录音。可从大量的细胞（最多200个），这是重要的，因为人类的精子可以在其自发的和刺激的Ca 2 +信号显示了很多的变化的响应。成功的标签，在录制过程中细胞的生存高度依赖对样品质量。可怜的样品给穷人标签，不良反应和细胞可能会死在录制过程中。 人类精子是非常敏感的光损伤，因此，我们用最低限度的，必要的照明（强度和曝光时间），以最大限度地提高细胞的存活和尽量减少由于细胞死亡的文物。一个高感光摄像头（允许使用低强度的照明）是大有裨益的，因为相机会拿起弱荧光。背照，EM - CCD相机特别适合，但非常昂贵。 LED照明（而不是使用一个氙汞灯激发滤光片也可提高细胞存活率（西垣等人，2006年）。 我们不使用FURA - 2，尽管比例度量成像的明显优势，因为FURA需要用紫外光激发，因为它是有必要采取两个图像，每个比例。对于单一波长，可见光染料，荧光强度正常化，主要为在细胞之间的染料加载差别进行补偿，但没有为单个细胞内的染料分布，这必须牢记获得的数据。虽然单一波长的染料，在原则上可以进行校准，技术的准确性差，而且我们不打算这样做。 而比例FURA - 2（或排放比染料印度）获得的数据，甚至无需校准，相对变化可以接受的忠实代表的[Ca 2 +] i的，单一波长的染料荧光强度远从线性相关的[Ca 2 +] i的在最有用的部分饱和曲线为单一波长染料的关系通常近似为对数。我们目前使用俄勒冈绿BAPTA - 1（图4），因为它是敏感的小变化的[Ca 2 +] i的接近静息水平（50-100纳米）。当的[Ca 2 +]我上升1微米以上的反应将荧光的变化和染料的代表性不足可能饱和。改善技术选项，而不诉诸紫外线激发双重负载细胞，如氟- 3和FURA红色染料。都可以被激发波长为488，但同时他们的反应是非常不同的。在540和650 nm处的排放记录提供了一个比它可以提供一个更好的方法，准确监测的[Ca 2 +] i的（Haughland 2002年），并可能适用于精子（Nisigaki等，2006 ）。 <img src="/files/ftp_upload/1996/1996fig4.jpg" alt="图4" /> 图4。俄勒冈绿BAPTA - 1和俄勒冈州的绿BAPTA - 2免费的[Ca 2 +（nm）和荧光发射峰值波长（约525纳米）之间的关系。 OGB1给出了一个较高的荧光在休息的[Ca 2 +]，但饱和在较低水平，从而有一个较小的可用范围。从分子探针手册（Haughland 2002年）获得这些地块的数据。

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<td>Low light level Camera</td>
<td>&nbsp;</td>
<td>QImaging</td>
<td>Rolera XR</td>
<td>&nbsp;</td>
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<td>Oregon Green BAPTA-1</td>
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<td>Invitrogen</td>
<td>06807</td>
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<td>Andor</td>
<td>IQ</td>
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<td>Image J</td>
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<td>Opto LED</td>
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<td>Pluronic F-127 (20% solution in DMSO)</td>
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techniques for imaging ca2+ signaling in human sperm

荧光显微镜观察细胞，用荧光，CA加载<sup&gt; 2 +</sup&gt;敏感的染料是用于CA的空间和时间方面的测量<sup&gt; 2 +</sup&gt;活细胞中的信号。在这里，我们描述了在我们的实验室中使用的方法，为人类精子悬浮加载CA<sup&gt; 2 +</sup报告染料和测量过程中的生理刺激的荧光信号。直接游泳运动型细胞分离和孵育0-24 h的capacitating条件下，取决于实验。细胞permeant上午（乙酰氧基甲基酯）的CA酯的形式<sup&gt; 2 +</sup&gt;报告染料，然后添加到一个细胞分装和1期H是允许进入细胞质染料加载。我们使用可见光波长染料，以尽量减少光损伤的细胞，但是这意味着，比例是无法进行录制。这种方法的优点和缺点进行了讨论。在装货期间细胞被引入到成像室，并坚持一个D -聚赖氨酸涂布盖玻片。在装载期间多余的染料和松散的细胞灌注仪器室的连接被删除。会议厅内连续灌注，刺激和修改salines的将被添加到灌注头。实验记录时间推移荧光图像采集，并通过手工绘图感兴趣区域，在详细脱机分析。数据标准化为刺激前的水平，这样，每个单元格（或一个单元格的一部分），图形显示CA<sup&gt; 2 +</sup&gt;％荧光变化的响应。

Watch this Scientific Journal Video about Techniques for Imaging Ca2+ Signaling in Human Sperm at JoVE.com

Techniques for Imaging Ca2+ Signaling in Human Sperm

刺激诱发的[Ca<sup&gt; 2 +</sup&gt;]：我个人的人类精子的信号进行评估。运动型细胞加载与Ca<sup&gt; 2 +</sup&gt;敏感的荧光染料（AM -酯法），并固定在perfusable室。时间推移荧光显微镜和细胞成像，通过灌注介质刺激。单细胞的反应（或地区）使用Excel进行离线分析。

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

techniques-for-imaging-ca2-signaling-in-human-sperm

Research

JoVE Journal

Biology

Techniques for Imaging Ca2+ Signaling in Human Sperm

17.0K 次观看.  University of Birmingham. 刺激诱发的[Ca 2 +]：我个人的人类精子的信号进行评估。运动型细胞加载与Ca 2 +敏感的荧光染料（AM -酯法），并固定在perfusable室。时间推移荧光显微镜和细胞成像，通过灌注介质刺激。单细胞的反应（或地区）使用Excel进行离线分析。

视频: Techniques for Imaging Ca2+ Signaling in Human Sperm

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

在体内的Ca 2 + -蜜蜂的嗅觉学习期间的蘑菇体神经元的成像

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

科研

生物学

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

荧光原位杂交（FISH）的议定书“在人类精子

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

血管钙的可视化 2 +信号旁分泌源性活性氧引发

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

一个高含量的成像流程研究Grb2的信号复合体的表达克隆

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

CA同时测量人的心房肌细胞分离<sup&gt; +</sup&gt;瞬变和膜电流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

测量细胞内Ca<sup&gt; +</sup在人类精子的变化采用4项技术：传统的荧光，停流荧光，流式细胞术及单细胞成像

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

成像前中期和减数分裂中期的我固定技术<em&gt;果蝇</em&gt;卵母细胞

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...