Name: measuring intracellular ca2+ changes in human sperm using four techniques: conventional fluorometry, stopped flow fluorometry, flow cytometry and single cell imaging
Uploaded: 2013-05-24T12:00:00
Description: Watch this Scientific Journal Video about Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell

作者感谢何塞·路易斯·德德拉维加，埃里卡Melchy和木村拓哉西垣博士的技术援助。这项工作得到全国理事会全国西恩西亚ŸTECNOLOGIA（国家科学技术委员会墨西哥）（99333和128566 CT）;局一般事务德尔个人Académico/国立大学自治墨西哥（IN202212-CT）。

在本文中，我们报道了使用上述四个技术来衡量的[Ca 2 +] i的变化在人类精子细胞。我们用孕激素引发的Ca 2 +响应，因为它是公认的，这种类固醇产生一个短暂的[的Ca 2 +] i的增加精子。特别是，在人精子，孕激素直接激活的Ca 2 +通道（即CatSper）表达于精子细胞的质膜10,11。我们还测量了休息的[Ca 2 +] i</su...

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细胞内的信号是至关重要的大多数细胞活动的Ca 2 +是一个无处不在的使者，伴随着哺乳动物细胞在其整个生命周期，从他们的起源在受精时，其生命周期结束。针对不同的刺激的[Ca 2 +] i的增加，振荡和录时空编纂;因此，多元化的进程被激活，调制或终止的Ca 2 +编码的消息。细胞内Ca 2 +动力学精子生理学是非常重要的，因为这涉及离子的信号转导过程中在一个复杂...

的Ca 2 +是一种通用的第二信使，在真核细胞中的信号转导通路。细胞内钙离子（Ca 2 +的一）参与许多基本的生理过程的调节兴奋和非兴奋性细胞。的重要性和普遍性的Ca 2 +作为第二信使的信号转导过程中的事件是来自于细胞内的信息在传输及其时空通用性。虽然的Ca 2 +可以不被合成的新生或退化内的细胞，其细胞内浓度（[钙2 +] i 的 ）是保持非常严格的限制内通过不同的细胞机制，不断缓冲，死骨，区域化，和/或累积的Ca 2 +。在该离子的浓度的变化可以发生在高度局部化区域内的电池1，解密这种波动是必不可少的获得解EPER（1）中的作用中的信令机制，（2）的生理意义，和（3）一般的细胞信号转导机制的理解。的Ca 2 +介导的信号转导是在第2精子生理特别重要的。精子活力受精成功的最重要的功能之一，而事实上，一些精子活动力缺陷可导致不育3-5。中的Ca 2 +鞭毛运动的重要性已确认6，但是如何的Ca 2 +鞭毛弯曲控制的具体形式，该机制尚不完全清楚。 在融合与卵子结合之前，精子必须经过获能，精子内居住的女性道依赖于一个复杂的过程。能过程中，精子膜的脂质结构和组织被修改，主要是由于从细胞膜的胆固醇去除。此外，一些蛋白质酪氨酸磷7 ylated 2。重要的是，能过程，增加细胞内pH值（pH值）的[Ca 2 +] i的，并在一些物种2的超极化膜电位。能的只需在亚精子（20-40％），在所有这些细胞的变化所涉及的机制远未清楚。人们普遍接受的，只有亚能精子发生顶体反应（AR），当暴露在生理电感。也是AR的Ca 2 +施肥在所有物种中具有专门的顶体（细胞器外层和内层膜）所需的受规管活动。在此过程中的外顶体膜与精子的质膜融合，释放的水解酶，使精子细胞渗透到周围的卵透明带（ZP）的糖蛋白质的基质。 AR还公开了一个新的膜融合与精子细胞表面为最后的两个配子融合蛋质膜。有几种细胞的配体诱导的AR，孕激素之一，其中研究最多的。 在这项工作中，我们提出了四种不同的技术涉及使用的Ca 2 +敏感的荧光染料来衡量的[Ca 2 +] i的变化，孕激素引发的人精子（除了流式细胞仪，我们测量了的[Ca 2 + ] i的增加诱导在体外获能过程）。在这种特殊情况下，我们用荧光凌晨3点（生命科技，大岛，NY），膜渗透性染料的K D = 325纳米， 在体外监测荧光变化作为时间的函数有三个方法，第四技术，我们测量了在一个单一的给定时间点的荧光值。这些不同的方法，相得益彰，因为他们完全提供了空间和时间的水库辨率在单细胞和细胞种群水平。 细胞群体或散装实验散装技术被广泛使用，不仅因为他们所需要的工具都一应俱全，而且还因为他们是简单，完善，并允许平均百万计的细胞进行测量，在一次实验中的信息。 技术＃1。传统的荧光 这种技术监测的荧光变化作为时间的函数的样品体积范围从200到1,000μl的玻璃比色皿中进行实验。加入试剂时，需要适当的混合磁力搅拌，因此，获得的时间分辨率是在以秒计。典型的细胞所分析的样品的浓度范围是10 5 -10 8个细胞/ ml。 技术＃2。停流荧光 Ť他的技术还监测作为时间的函数的荧光的变化，但迅速混合在一起，此试剂包含一个非常小的样品体积（从25-100微升）到记录比色皿（使用压力）。因此，均质化试剂是瞬时的，能够在毫秒级的时间分辨率高。分析所得到的荧光随时间的痕迹是适合用于确定反应速率，阐明反应机理的复杂性，常见的细胞所分析的样品的浓度范围内获得上短命的反应中间体等的信息是10 5 -10 7细胞/ ml。 单细胞实验批量实验报告了大量的单元格的平均行为，但是，人口可能经常表现出这种类型的测量过程中不受干扰的异构属性。因此，单细胞技术用于补充日E信息的细胞群体的实验获得。 技术＃3。流式细胞仪 尽管从单细胞测量的重要性，所产生的信息，重要的是要分析大量的单元格，以防止错误的推断小区固有属性的整个人口。出于这个原因，高通量技术的青睐和最常用的方法是流式细胞仪，其中10,000个细胞常规分析每个条件。此方法使不同的人群多参数分析细胞分类根据它们的大小（前向散射（FSC）），粒度（侧向散射光（SSC））和荧光强度（与抗体的特异性标记，生存能力的盯防，等） ，从而提供了参数的分布的信息的一组单元格。流式细胞仪提供即时的，而不是随时间变化的信息8。正向和侧向散射光值ARE也可用于选择一个栅极，包括细胞，但对于测量荧光，阴性和阳性荧光控制判别的细胞碎片，灰尘等也必须被包括在内。如果使用一个以上的荧光通道，这个过程被称为补偿必须执行（详见<a href="http://www.bdbiosciences.com/resources/protocols/setting_compensation.jsp" target="_blank">http://www.bdbiosciences.com/resources/protocols/setting_compensation.jsp</a> ）。补偿允许为光谱重叠荧光团之间的歧视。流式细胞术还允许歧视的死细胞，一般通过碘化丙啶（PI）染色。 技术＃4。单细胞成像 显微镜是另一种常见的方法，研究单细胞的行为，它非常适合于随时间变化的研究，而且还提供了空间分辨率。一个主要缺点是高通量分析仅处于起步阶段，在目前的时间<s高达9。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Ham's F-10</td>
 <td>Sigma-Aldrich</td>
 <td>N-6013</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Sigma-Aldrich</td>
 <td>A-7906</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium Chloride Dihydrate approx. 99%</td>
 <td>Sigma-Aldrich</td>
 <td>C-3881</td>
 <td></td>
 </tr>
 <tr>
 <td>Makler Counting Chamber</td>
 <td>SEFI Medical Insruments LTD</td>
 <td>SEF-MAKL</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluo-3 AM</td>
 <td>Invitrogen</td>
 <td>F-1242</td>
 <td>20 vials/50 &mu;g each</td>
 </tr>
 <tr>
 <td>Ionomycin</td>
 <td>Alomone</td>
 <td>I-700</td>
 <td></td>
 </tr>
 <tr>
 <td>Progesterone</td>
 <td>Sigma-Aldrich</td>
 <td>P0130</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>S-9888</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Potassium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>P-3911</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Sodium bicarbonate</td>
 <td>JT Baker</td>
 <td>3506</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Magnesium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>M-2670</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Calcium chloride anhydrous</td>
 <td>Sigma-Aldrich</td>
 <td>C-1016</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H-3125</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>D-Glucose</td>
 <td>JT Baker</td>
 <td>1906-01</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Sodium pyruvate</td>
 <td>Sigma-Aldrich</td>
 <td>P-2256</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Sodium L-lactate (aprox. 99%)</td>
 <td>Sigma-Aldrich</td>
 <td>L- 7022</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Propidium Iodide </td>
 <td>Invitrogen</td>
 <td>L-7011 </td>
 <td>Component B</td>
 </tr>
 <tr>
 <td>Triton X-100 (t-Octylphenoxypolyethoxyethanol)</td>
 <td>Sigma- Aldrich</td>
 <td>X-100</td>
 <td>2.4 mM solution in water </td>
 </tr>
 <tr>
 <td>Round coverslip </td>
 <td>VWR</td>
 <td>48380 080</td>
 <td>25 mm diameter</td>
 </tr>
 <tr>
 <td>Poly-L-lysine solution </td>
 <td>Sigma-Aldrich</td>
 <td>P8920</td>
 <td></td>
 </tr>
 <tr>
 <td>Manganese chloride</td>
 <td>Sigma-Aldrich</td>
 <td>M-3634</td>
 <td></td>
 </tr>
 <tr>
 <td>Attofluor; Cell Chamber, for microscopy</td>
 <td>Life technologies</td>
 <td>A-7816</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethyl Sulphoxide</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>5x5 ml</td>
 </tr>
 </tbody>
</table>

measuring intracellular ca2+ changes in human sperm using four techniques: conventional fluorometry, stopped flow fluorometry, flow cytometry and single cell imaging

精子是男性生殖细胞特别设计，以达到与卵子结合，识别和融合。要执行这些任务，精子细胞必须准备好面对不断变化的环境和来克服一些物理障碍。在本质上转录和翻译默默的，这些运动细胞的深刻依赖不同的信号机制，将自己定位和定向的方式游泳，具有挑战性的环境条件，在旅途中找到鸡蛋抗衡。特别的Ca 2 +介导的信号是关键的几个精子功能：激活的运动，获能和顶体反应（胞吐的事件，使精卵融合）（一个复杂的过程，精子顶体反应准备）。使用荧光染料来跟踪该离子的细胞内波动是由于它们易于应用，灵敏度显着的重要性，和多功能性DET挠度。使用单一染料加载协议，我们使用四种不同的荧光技术监测精子的Ca 2 +的动态。每种技术都提供了不同的信息，使空间和/或时间分辨率，在单细胞和细胞种群水平产生数据。

<img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50344/50344fig1highres.jpg" src="/files/ftp_upload/50344/50344fig1.jpg" /> 图1。示意图实验的协议精子样品制备游泳方法。活动精子分离，并调整其浓度的主要步骤进行了说明。最后孵化步骤只获能需要时执行。 <img alt="图2" src="/files/ftp_upload/50344/50344fig2.jpg" fo:content-width=...

Watch this Scientific Journal Video about Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell 

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

细胞内Ca<sup&gt; +</sup&gt;动力学是非常重要的，在精子生理学和Ca<sup&gt; +</sup&gt;敏感的荧光染料构成一个多功能的工具来研究它们。人口实验（荧光和停流荧光）和单细胞实验（流式细胞仪和单细胞成像）是用来跟踪时空的[Ca<sup&gt; +</sup&gt;]在人类精子细胞中的变化。

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 ...

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

Techniques for Imaging Ca2+ Signaling in Human Sperm

Techniques for Imaging Ca2+ Signaling in Human Sperm

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 ...

科研

生物学

Flow Cytometry Purification of Mouse Meiotic Cells

流动小鼠减数分裂细胞的流式细胞仪净化

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

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

代谢标记和流式细胞仪测定细胞周期进程的动力学

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

该<em&gt;爪蟾</em&gt;卵母细胞切割开凡士林隙电压钳技术与荧光

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

LED热流量 - 结合光遗传学与流式细胞仪

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

通过使用多光谱成像流式细胞术测量LC3，p62和LAMP1共定位评估自噬通量

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

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