我们感谢 AQUAGAMETE 1205 的成本行动。这项工作得到了 AGL201568330-C2-1-R 项目 (MINECO/菲德) 的财政支持。大卫 g. Valcarce 由 EDU1084/2012 和 Fondo 社会 Europeo 资助。作者承认安娜 Riaza 博士和 Stolt 海洋农场, Paulino 博士, 拉巴斯博士蒙特罗和何塞拉蒙 Guiérrez。我们还感谢宝拉. 费尔南德斯. Colado 为录像。

注: Fluorochrome 孵化和共焦分析将采取至少2-3 小时的控制和治疗样品。在这个时间计算中不包括数据处理。所需材料可在材料表中找到。本协议可应用于新鲜或冷冻保存的精子。Solea senegalensis是一种在冷水中产生的鱼类, 通常在寒冷的环境下工作 (4-7 摄氏度)。有关协议的一般视图, 请参见图 1 。
1. 实验前的准备工作
<ol><li>在分析级二甲基亚砜上制备1毫米线粒体染色液。</li>
	<li>在去离子水中制备1微克/毫升 4′-, 6-diamidino 2-苯基吲哚盐酸盐 (DAPI) 溶液。</li>
	<li>准备一个 200 mOsm/千克响铃解决方案 (116 毫米氯化钠, 2.9 毫米氯化钾, 1.8 毫米 CaCl2, 5 毫米 HEPES, pH 7.7)</li>
	<li>在分析级甲醇上制备4毫米-2′-、7′-dichlorodihydrofluorescein 双乙酸 (DCFH DA) 溶液。 注: 保持库存解决方案在整除数-20 摄氏度, 直到需要。</li>
	<li>将离心设置为4-7 摄氏度。</li>
</ol>2. Fluorochrome 孵化前的样品准备
注意: 当吹打和 resuspending 时, 对精子的温和处理是可取的。
<ol><li>如果使用保存的细胞, 准备一个水浴为解冻在40°c 为Solea senegalensis精子10。将秸秆样品浸入水浴中7秒。</li>
	<li>使用剪刀切割, 把样品空成一个离心管, 离心机在 1000 x g, 4-7 摄氏度为1分钟。</li>
	<li>用吹打从冷冻保存的样品中取出精浆或精浆冷冻保护剂, 丢弃任何可能干扰染色协议的成分。</li>
	<li>并用重悬细胞在 50-100 ul 4 °c 200 mOsm/公斤响铃解决方案。</li>
	<li>用 Neubauer 或类似的计数室在立体显微镜下测定精子浓度。</li>
	<li>将细胞悬浮液稀释至 1-2 x 106细胞/mL, 在4摄氏度的溶液中最终体积为0.5 毫升。</li>
</ol>3. Fluorochrome 孵化
注: 荧光必须在低光照条件下处理, 特别是在溶液中。
<ol><li>添加 3.125 ul DCFH-DA 股票溶液 (最后浓度在样品:25 微米) 到工作样品稀释。在黑暗中孵化4-7 摄氏度40分钟 (10。</li>
	<li>在30分钟以后, 增加 0.5 ul DAPI 存货解答 (最后的集中在样品: 2 微米) 和 0.5 ul 线粒体污点存货解答 (最后的集中在样品: 100 毫微米) 对样品。在黑暗中孵化两荧光10分钟。</li>
</ol>4. 共焦显微术的样品准备
<ol><li>潜伏期后, 离心机的细胞悬浮在 1000 x g, 4-7 摄氏度为1分钟。</li>
	<li>吹打小心地丢弃上清。</li>
	<li>添加 10-20 ul 的 200 mOsm/公斤的铃声解决方案。</li>
	<li>放置 5 ul 的浓缩细胞悬浮下降在幻灯片上。</li>
	<li>小心, 把封面幻灯片上的准备, 并密封它与波兰。一旦波兰人干了, 准备准备为共焦显微镜。</li>
</ol>5. 实验前共聚焦设置
注: 根据显微镜, DCFH 可能被 "烧伤"。先用非贵重样品建立最佳条件。
<ol><li>打开显微镜、激光、照相机和运行显微镜的电脑。</li>
	<li>设置励磁激光器, 考虑到每个 fluorochrome 的激发和发射最大度:<ol>
<li>对于 DAPI, 使用的激发最大值为 358 nm, 发射最大值为 465 nm。</li>
		<li>对于线粒体染色, 使用最多 644 nm 的激发和发射最大值为 662 nm。</li>
		<li>对于 DCFH, 使用的激发最大值为 504 nm, 发射最大值为 525 nm。</li>
	</ol>
</li>
	<li>开始使用25X 目标, 专注于精子。选择要集中的 DAPI 通道, 因为此 fluorochrome 报告的强度最高。一旦单元格集中, 请使用63X 或100X 目标进行彻底评估, 因为Solea senegalensis精子大小约为2微米。</li>
	<li>对于每个通道, 优化针孔, 增益和电压。同样地, 调整数字偏移量以减少背景。为所使用的 fluorochrome 优化所有参数后, 保存设置。对于单个Solea senegalensis精子细胞的常规实验, 我们使用了以下方法 (这些参数可能会在每个实验中发生变化): 针孔1和增益电压 750 v。</li>
</ol>6. 图像的采集
<ol><li>为图像获取选择所需的质量条件。定义采集设置, 如钻头深度, 图像格式, 光片厚度, 并选择单面照明。对于单个Solea senegalensis精子的常规实验, 我们使用了以下方法 (这些参数可能在每个实验中发生变化): 1024年像素的帧大小;位每像素 16;扫描速度为 2-4)。</li>
	<li>打开并居中扫描区域以开始并采取该字段的图像。</li>
	<li>使用裁剪工具, 选择感兴趣的区域并按 "实时"。</li>
	<li>在 "范围指示器" 工具的帮助下, 调整数字偏移量以减少背景。为每个通道执行此项, 并采取该图像。</li>
	<li>检查每个 fluorochrome 及其组合的各个通道的质量。</li>
</ol>7. 创建3D 图像视频
<ol><li>Z 堆栈捕获设置。<ol>
<li>在软件中选择 Z 堆栈选项。</li>
		<li>选择单个单元格、一组单元格或感兴趣的区域并将其聚焦。</li>
		<li>使用 "第一个切片" 和 "最后一个切片" 选项分隔 z 堆栈, 并在精细焦点的帮助下, 将 z 步骤设置为间隔为0.2 µm。选择 DAPI 频道集中, 因为这 fluorochrome 可能报告的最高强度, 你可以很容易地选择整个精子头。选择每个通道的最佳采集参数并运行实验。</li>
	</ol>
</li>
	<li>运行 Z 堆栈实验。拆分通道并观察单元格内每个信号的定位。</li>
	<li>Z 堆栈过程。<ol>
<li>卷渲染: 实验完成后, 选择 Z 堆栈处理软件的选项, 然后选择 "音量渲染" 选项。选择步骤数和旋转度数 (360°)。使用视频扩展名保存文件。</li>
		<li>立体声浮雕: 在 "选项" 过程窗口中选择 "立体声浮雕"。此工具允许使用分割通道创建3D 图像视频。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Amaral, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+functionality+and+chemical+compound+action+on+sperm+function.">Mitochondrial functionality and chemical compound action on sperm function.</a> Curr Med Chem. , (2016).</li><li>Morielli, T., O'Flaherty, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+impairs+function+and+increases+redox+protein+modifications+in+human+spermatozoa.">Oxidative stress impairs function and increases redox protein modifications in human spermatozoa.</a> Reproduction. 149 (1), 113-123 (2015).</li><li>G&#252;rler, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+sperm+viability,+synthesis+of+reactive+oxygen+species,+and+DNA+damage+of+bovine+sperm.">Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.</a> Theriogenology. , (2016).</li><li>Zhu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+E+Analogue+Improves+Rabbit+Sperm+Quality+during+the+Process+of+Cryopreservation+through+Its+Antioxidative+Action.">Vitamin E Analogue Improves Rabbit Sperm Quality during the Process of Cryopreservation through Its Antioxidative Action.</a> PLoS One. 10 (12), e0145383 (2015).</li><li>Fang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+ROS+production+through+mitochondria-targeted+antioxidant+and+mitochondrial+uncoupling+increases+post-thaw+sperm+viability+in+yellow+catfish.">Inhibition of ROS production through mitochondria-targeted antioxidant and mitochondrial uncoupling increases post-thaw sperm viability in yellow catfish.</a> Cryobiology. 69 (3), (2014).</li><li>Thomson, L. K., Fleming, S. D., Aitken, R. J., De Iuliis, G. N., Zieschang, J. A., Clark, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation-induced+human+sperm+DNA+damage+is+predominantly+mediated+by+oxidative+stress+rather+than+apoptosis.">Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis.</a> Hum Reprod. 24 (9), 2061-2070 (2009).</li><li>Kim, S. H., Yu, D. H., Kim, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+phosphatidylserine+translocation,+intracellular+hydrogen+peroxide,+and+DNA+integrity+in+canine+sperm.">Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm.</a> Theriogenology. 73 (3), (2010).</li><li>Guthrie, H. D., Welch, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reactive+oxygen+species+on+sperm+function.">Effects of reactive oxygen species on sperm function.</a> Theriogenology. 78 (8), 1700-1708 (2012).</li><li>Aitken, R. J., Jones, K. T., Robertson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+and+sperm+function--in+sickness+and+in+health.">Reactive oxygen species and sperm function--in sickness and in health.</a> J Androl. 33 (6), (2012).</li><li>Valcarce, D. G., Robles, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+captivity+and+cryopreservation+on+ROS+production+in+Solea+senegalensis+spermatozoa.">Effect of captivity and cryopreservation on ROS production in Solea senegalensis spermatozoa.</a> Reproduction. 152 (5), (2016).</li><li>Gibb, Z., Lambourne, S. R., Aitken, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+paradoxical+relationship+between+stallion+fertility+and+oxidative+stress.">The paradoxical relationship between stallion fertility and oxidative stress.</a> Biol Reprod. 91 (3), (2014).</li><li>Cabrita, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+enhancing+fish+sperm+quality+and+emerging+tools+for+sperm+analysis.">Factors enhancing fish sperm quality and emerging tools for sperm analysis.</a> Aquaculture. 432, 389-401 (2014).</li></ol>

作者没有什么可透露的。

众所周知, 线粒体是精子运动和功能的关键细胞器。这些细胞器同时直接参与 ROS 的生产。有趣的是, 适当的精子功能需要控制的活性氧含量为1。在哺乳动物中, 生育率和氧化应激之间的正相关关系已显示在11中, 但过度的水平影响精子质量12。一个关键的因素, 可能是决定性的积极或消极的影响, 不仅是 ros 水平, 而且 ros 细胞内定位。ros 的有害?...

活性氧种类的生产已与精子质量最近的1。虽然线粒体中的 ros 生产可以被认为是正常的生理过程, 但氧化应激是由过量的 ros 产生的, 是导致精子在不同水平上损伤的明显原因。在人类中, 氧化应激与男性不育、改变运动和接受精子2的能力有关;在哺乳动物中, 冷冻精子样品中 DNA 完整性的变化也与 H2O23的合成有关。
冷冻保存是水产养殖中的一种常用的基因银行技术。这种技术在具有生殖问题 (如Solea senegalensis) 的物种中尤为重要。这个市场上有价值的物种显示, 由于缺乏求爱, 在圈养的个体中繁殖功能失调。这一事实使精子的超低温保存必须有精子可用性的人工受精。然而, 冷冻保存可能是氧化应激的来源, 可能是有害的精子4 , 因为研究报告了有益的效果, 补充抗氧化剂。据报道, 通过线粒体靶向抗氧化剂的活性氧抑制有助于黄鲶鱼的精子超低温保存5。
因此, 精子样本中 ROS 的含量是很重要的, 特别是在保存6、7之后, 因为这些分子被认为是精子生存和生育能力8的一个缺点。此外, 研究细胞中 ROS 的分布对于推断潜在损伤的程度是至关重要的。例如, 线粒体中的低活性氧能被认为是正常的, 与精子功能相容, 但细胞核内的高活性氧含量可能是精子 DNA 损伤的指标。H2O2是最相关的 ROS 之一, 可以从线粒体中释放出来, 穿透细胞核, 因为它是一个小而无电荷的分子9。Dichlorofluorescein 双乙酸 (DCFH) 可以明确揭示胞内过氧化物发光的绿色荧光。本文介绍了用共聚焦显微镜检测Solea senegalensis精子中 H2O2细胞内定位的详细协议。

<table><tbody><tr><td>2&amp;prime;,7&amp;prime;-Dichlorodihydrofluorescein diacetate (DCFH-DA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D6883</td><td></td></tr><tr><td>4&amp;prime;,6-diamidino-2-phenylindole (DAPI)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C1016&amp;nbsp;</td><td></td></tr><tr><td>Confocal Microscopy</td><td>Zeiss</td><td>LSM800</td><td></td></tr><tr><td>Cover slips</td><td>Thermo Fisher Scientific</td><td>12-541B</td><td></td></tr><tr><td>DMSO, Analytical Grade</td><td>Sigma-Aldrich</td><td>W387520</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>Methanol, Analytical Grade</td><td>Sigma-Aldrich</td><td>34860</td><td></td></tr><tr><td>MitoTrackerDeep Red&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>M22426</td><td></td></tr><tr><td>Microcentrifuge (refrigerated)</td><td>Thermo Fisher Scientific</td><td>75002441</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653&amp;nbsp;</td><td></td></tr><tr><td>Neubauerchamber</td><td>Sigma-Aldrich</td><td>BR717810</td><td></td></tr><tr><td>Slides</td><td>Thermo Fisher Scientific</td><td>10143562BEF</td><td></td></tr></tbody></table>

evaluation of intracellular location of reactive oxygen species in solea senegalensis spermatozoa

氧化应激是降低精子质量的重要因素之一。开发有效的检测精子中活性氧 (ROS) 的协议在任何物种中都是非常重要的, 但是这些方法在硬骨鱼类中很少使用, 甚至更少。冷冻保存是水产养殖的一种有用的技术, 为不同的目的, 包括基因银行和保证精子的可用性全年。冷冻/解冻程序可能导致 ROS 的产生和损伤精子细胞。考虑到过量的 ROS 生产可能导致精子的潜在损害取决于它们的定位, 这里有一个详细的方法来检测 H2O2和评估其细胞内定位的共焦显微镜提供。为此, 采用3荧光 (-2′-、7′-Dichlorodihydrofluorescein 双乙酸 (DCFH DA)、活线粒体染色和4′-、6-Diamidino-2-苯基吲哚二盐酸盐 (DAPI)) 来评价H2 O 2 的协同定位与精子细胞核或线粒体在Solea senegalesis精子样本。

共焦显微术是硬骨鱼类精子细胞内 ROS 评价的理想方法。这项研究 (图 1) 中提出的三荧光 (DAPI、线粒体染色和 DCFH) 的组合提供了许多有用的信息, 可应用于基础研究, 并可应用于改进工业水产养殖植物, 如冷冻保存协议。可以进行不同类型的分析, 以关联细胞内 ROS 的存在和其他参数: 运动, 生存能力, 不同模式之间的良好和不良育种, 活化运动或季节性?...

Watch this Scientific Journal Video about Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa at JoVE.com

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

此协议描述了一个用于检测Solea senegalensis精子中的 H2O2本地化的详细方法, 使用敏感的 fluorochrome DCFH-DA 为 ROS、线粒体的活线粒体染色和细胞核的 DAPI可视化, 分别。该协议的设计是在2小时内进行, 无论是新鲜或解冻精子。

Solea Senegalensis精子中活性氧的细胞内定位评价

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) ...

evaluation-intracellular-location-reactive-oxygen-species-solea

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

6.2K 次观看.  University of León. 此协议描述了一个用于检测Solea senegalensis精子中的 H2O2本地化的详细方法, 使用敏感的 fluorochrome DCFH-DA 为 ROS、线粒体的活线粒体染色和细胞核的 DAPI可视化, 分别。该协议的设计是在2小时内进行, 无论是新鲜或解冻精子。

视频: Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

科研

生物化学

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes require energy. There remains an ongoing debate about the sources generating the ATP which fuels sperm progressive motility, capacitation, hyperactivation, and acrosome reaction. Here, we describe the application of an extracellular flux analyzer as a tool to analyze changes in energy metabolism during mouse sperm capacitation. Using H+- and O2- sensitive fluorophores, this method allows monitoring glycolysis and oxidative phosphorylation in real-time in non-capacitated versus capacitating sperm. Using this assay in the presence of different energy substrates and/or pharmacological activators and/or inhibitors can provide important insights into the contribution of different metabolic pathways and the intersection between signaling cascades and metabolism during sperm capacitation.

We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation.

使用细胞外通量分析仪测量小鼠精子发作期间糖解和氧化磷酸化的变化

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...

Fluorimetric Techniques for the Assessment of Sperm Membranes

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4&#39;,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

精子膜评价的荧光技术

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, ...

发育生物学

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

检测，可视化和定量活性氧的生成在变形虫模型系统

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

生物学

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

使用2'，7'-二氯荧光素双乙酸探针和流式细胞术在穆勒神经胶质细胞中定量活性氧

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

神经科学

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility.
With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 &#176;C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5&#8242;,6,6&#8242;-tetrachloro-1,1&#8242;,3,3&#8242;-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.

The present protocol provides a practical solution that allows for the measurement of apoptosis, mitochondrial membrane potential, and DNA damage in human sperm with a single cytometer.

用于检测人类精子功能缺陷的生物标志物的流式细胞术分析

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no ...

医学

通过高分辨率呼吸测定法测定人精子细胞中的呼吸

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm mitochondrial activity, so the measurement of sperm mitochondrial function is an indicator of sperm quality. High-resolution respirometry is a method of measuring the oxygen consumption of cells or tissues in a closed-chamber system. This technique can be implemented to measure respiration in human sperm and provides information about the quality and integrity of the sperm mitochondria. High-resolution respirometry allows the cells to move freely, which is an a priori advantage in the case of sperm. This technique can be applied with intact or permeabilized spermatozoa and allows for the study of intact sperm mitochondrial function and the activity of individual respiratory chain complexes. The high-resolution oxygraph instrument uses sensors to measure the oxygen concentration coupled with sensitive software to calculate the oxygen consumption. The data are used to calculate respiratory indices based on the oxygen consumption ratios. Consequently, the indices are the proportions of two oxygen consumption rates and are internally normalized to the cell number or protein mass. The respiratory indices are an indicator of sperm mitochondrial function and dysfunction.

作者聚焦:通过揭示精子代谢和线粒体功能推进男性不育研究 

The analysis of sperm mitochondrial function by high-resolution respirometry permits the measurement of the oxygen consumption of freely moving spermatozoa in a closed-chamber system. The technique can be applied to measure respiration in human spermatozoa, which provides information on sperm mitochondrial characteristics and integrity.

高分辨率呼吸测定法评估人类精子的线粒体功能

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm ...

Solea Senegalensis精子中活性氧的细胞内定位评价

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此视频中的章节

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

使用细胞外通量分析仪测量小鼠精子发作期间糖解和氧化磷酸化的变化

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

精子膜评价的荧光技术

检测，可视化和定量活性氧的生成在变形虫模型系统

在斑马鱼胚胎氧化应激分析

使用2'，7'-二氯荧光素双乙酸探针和流式细胞术在穆勒神经胶质细胞中定量活性氧

用于检测人类精子功能缺陷的生物标志物的流式细胞术分析

高分辨率呼吸测定法评估人类精子的线粒体功能

高分辨率呼吸测定法评估人类精子的线粒体功能