Name: quantitative immunofluorescence to measure global localized translation
Uploaded: 2017-08-22T13:00:00.000+00:00
Duration: 9 min 13 s
Description: Watch this Scientific Journal Video about Quantitative Immunofluorescence to Measure Global Localized Translation at JoVE.com

我们感谢这份手稿的批判性解读博士 Rachid Mazroui （拉瓦尔大学、 加拿大魁北克省）。我们感谢它们的技术援助研究中心细胞成像单元。M.法国霍是全宗德初中 1 研究学者研究杜魁北克-健康 (FRQ S)。这项工作被支持由加拿大卫生研究院 （授权号卫生研究院，拖把 286437 到 M.É。霍）。

1。 Puromycilation 条件测定 注: 这种技术描述了用于评估期间 MRC 5 细胞粘附过程 5 本地化的翻译的方法。Puromycilation 可以做任何单元格中，是重要的是优化 puromycilation 条件为特定的单元格行使用，因为治疗条件是不相同的嘌呤霉素浓度和所需的每个单元格行孵育时间。若要显示这些条件如何定义，三个示例细胞株 （即， HeLa、 MRC-5 和 Huh 7） 患者的嘌呤浓度增加为不同孵育时间 ( 图 1)。
<ol><li>成长相同数字的细胞在 24 孔板在 0.5 毫升的适当完成培养基在考试前 16 h。30,000 种子 MRC 5 单元格、 50,000 HeLa 细胞或 50,000 HuH 7 细胞每口井。一定要避免超过 60-70%汇合处 (图 1A)。</li>
	<li>准备 6 管的管 1.5 毫升的完整的细胞培养液中的嘌呤霉素 （0、 5、 10、 15、 20、 和 30 微克/毫升） 的浓度增加。预暖在 37 ° C</li>
	<li>每一集中的嘌呤霉素介质 （准备在步骤 1.2），转让从 0.5 毫升每到 6 新管管并将放线菌酮在终浓度为 50 微克/毫升添加到所有 6 新管。预暖在 37 ° C</li>
	<li>更改具有 0.5 毫升的完全培养基 （行 1 和 2） 的介质。对于第三行，添加 0.5 毫升的完整文化培养基用 50 微克/毫升的放线菌酮 ( 图 1B)。 
	注: 放线菌酮治疗已成为蛋白质 puromycilation，因为它能够阻止翻译伸长的最佳消极控制。因为嘌呤霉素纳入需要翻译伸长，放线菌酮治疗导致损失 puromycilated 蛋白信号 5 ， 18。</li>
	<li>孵育 15 分钟在 37 ° C (5%CO 2)。</li>
	<li>添加 0.5 毫升的准备步骤 1.2 到第二行中以获得最终浓度 0，2.5，5，7.5，10 和 15 微克/毫升，分别列 A、 B、 C、 D、 E 和 F.嘌呤霉素介质同时，添加 0.5 毫升的嘌呤霉素放线菌酮补充介质 （步骤 1.3） 在第三行 ( 图 1)。</li>
	<li>孵育 5 分钟在 37 ° C (5%CO 2)。</li>
	<li>添加 0.5 毫升的嘌呤霉素介质制备步骤 1.2 到第一行中获得最终浓度 0.0，2.5，5，7.5，10 和 15 微克/毫升 ( 图 1)。</li>
	<li>孵育 5 分钟在 37 ° C (5%CO 2)。</li>
	<li>洗每好从行 1 至 3 与 1 毫升的冰冷 1 X 磷酸盐缓冲液 (PBS) 5 分钟后加入的嘌呤霉素对井的第一行 （洗两次）。添加 1 X Laemmli 缓冲区的 75 微升 (4%十二烷基硫酸钠 (SDS)、 2-巯基乙醇 4%、 0.120 M 三 HCl pH 6.8，0.004%溴酚蓝和 10%的甘油) 来获得对每个条件的全细胞裂解液。</li>
	<li>运行 10 %sds-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 使用 1/3 的制备好的样品，以评估嘌呤霉素纳入。
	<ol><li>确定的嘌呤霉素纳入由印迹分析使用反嘌呤霉素抗体 (12 D 10) 稀释比率为 1: 25000 在原发性抗体孵化缓冲区 （2%牛血清白蛋白 (BSA)）、 430 毫米氯化钠、 10 毫米三 pH 7.4 和 0.01%的水平叠氮化钠）。 
		注: 对应于不同的单元格行提取物在步骤 1 中所述的代表图像显示为 图 2 中的例子。</li>
	</ol></li></ol> 2。在 Cellulo本地化翻译可视化 注: 此处描述的技术被用来评估本地化的翻译内结果 MRC 5 细胞粘附过程 5 期间。虽然在这里使用 MRC 5 单元格，其他细胞系可以用同样的方法步骤 2.1 中所述。
<ol><li>细胞制备。
	<ol><li>分离 MRC 5 细胞在汉克使用 0.25%胰蛋白酶/2.21 毫米乙二胺四乙酸 (EDTA) &#39; s 平衡盐溶液 (HBSS)。颗粒细胞在 15 毫升锥形离心管中离心 （200 x g 5 分钟），并暂停在杜尔贝科 &#39; s 改良鹰培养基 (DMEM) 含 10%胎牛血清 (FBS) 40,000 细胞/ml 后计数与计数室。</li>
		<li>孵育的悬浮的细胞，在 37 ° C 下使用 20 分钟的管转子保持他们在悬浮液中的温柔旋转。 
		注意: 这一步是关键，以便粘着复合物的完整分离。</li>
		<li>板 2 毫升的悬浮 MRC 5 细胞 （40,000 细胞/毫升） 在两个 35 毫米玻璃底部菜。Puromycilation 测定使用 35 毫米玻璃底部第一道菜 （请参见步骤 2.1.5） 和使用第二个作为阴性对照 （请参见步骤 2.1.6）。</li>
		<li>使细胞在 37 ° C (5%CO 2) 55 分钟，以便细胞粘附和碳化硅形成。</li>
		<li>添加嘌呤霉素使用浓度先前在第 1 节中定义的 35 毫米玻璃-底菜 （检测） 的载体。治疗 MRC 5 细胞，在 37 ° C (5%CO 2) 5 分钟使用 10 微克/毫升嘌呤霉素。</li>
		<li>作为阴性对照，放线菌酮在第二个 35 毫米玻璃底部菜 40 分钟后添加种子细胞前对待他们的 50 微克/毫升的放线菌酮 ( 图 2B)。然后，使用相同的浓度和孵化时间与步骤 （例如， 使用 10 微克/毫升的嘌呤霉素在 37 ° C (5%CO 2) MRC 5 5 分钟） 2.1.5 培养基加嘌呤霉素放线菌酮加后 15 分钟。</li>
		<li>洗两次的冰冷的 2 毫升每板 1 X PBS 和修复细胞与 1 毫升的 4%甲醛 （在 1 X PBS 稀释） 15 分钟在室温 (RT)。 
		注意: 多聚甲醛必须严格在使用化学罩。</li>
	</ol></li> <li>免疫组化。
	<ol><li>多聚甲醛固定后, 三次用 2 毫升的 1 X PBS 洗和在 500 µ L 的 PBS-TritonX-100 （0.5%) 孵育 20 分钟在室温下以通透细胞细胞。</li>
		<li>为了防止非特异性抗体结合，阻止样品用 500 µ L PBS &#8211; 牛血清白蛋白 （1%) 室温 20 分钟</li>
		<li>洗三次以 2 毫升的 PBS-Tween20 （0.1%) 和孵育 300 µ L 的稀释的抗体抗嘌呤霉素 (12 D 10) 1:12,500 在 1 X PBS 为 1 h 在室温下。</li>
		<li>洗 3 次用 PBS-Tween20 （0.1%) 2 毫升和孵育的抗鼠 IgG 共轭对荧光 300 微升 (这里，488 nm) 稀释在 PBS 可视化 puromycilated 多肽与鬼笔共轭对另一种荧光 (这里，555 nm)可视化 F-肌动蛋白。孵育 1 h 室温</li>
		<li>洗三次以 2 毫升的 PBS-Tween20 （0.1%)，然后孵育 5 分钟在室温下在 300 µ L 的 PBS-Tween20 （0.1%) 辅以 1 微克/毫升 4 &#39;，6-脒-2-苯基吲哚 (DAPI)。</li>
		<li>洗净用 2 毫升的 PBS-Tween20 （0.1%) 的三倍，然后用 2 毫升的 1 X PBS 清洗。通过在图像采集过程中保持 2 毫升 1 X PBS 的样品干燥防止细胞。</li>
	</ol></li></ol> 3。免疫荧光图像采集 注: 以下方法可用于任何商业上可用的共焦成像系统.
<ol><li>确定适当设置每个荧光定量的动态范围最大化。 
	注: 如果避免像素饱和度和信号阈值作适当的调整，则只可以获得代表量化。这些设置可以达到通过仔细调整激光功率，高电压 (HV)，获得，和偏移量。
	<ol><li>调整缩放因子 (5 X) 和像素 (512 × 512) 像素进行尺寸优化，数目。 
		注: 这应该是至少 2.3 倍小于光学分辨率，根据奈奎斯特定理。</li>
		<li>降低扫描速度 （12.5-20 微秒/像素），使用平均 （2-3 倍） 来改善信号的信噪比根据上文所述的设置。</li>
	</ol></li> <li>手动确定适当的顶部和底部焦平面沿 z 轴与最优步长 （0.4 微米/切片）。 
	注: 一步大小飞机是由轴向分辨率除以 2.3，根据奈奎斯特定理确定。通常情况下，20-25 层，以覆盖整个单元厚度的贴壁细胞。</li>
	<li>获得 60 X 计划载脂蛋白油浸物镜 （1.42 数值孔径） 整个单一单元共聚焦显微图像。</li>
</ol> 4。免疫荧光图像量化 <ol><li>富集测定。
	<ol><li>打开一个图像对应于 z 平面层的兴趣从获取单元格数据文件使用 ImageJ 软件 （可用的免费软件在 http://fiji.sc）。</li>
		<li>使用绘制直线线条绘图工具 (工具条 &#8594; 直) 通过量化那里所需的单元格区域。通过双击修改线宽 &#34; 直; &#34; 强度平均值通过 （在 图 3 中的 8 处设置） 线条的宽度。 
		注: 一些细线条首选，避免了不同结构的信号重叠。</li>
		<li>后正确地设置的线，配置文件确定轴信号密度 (菜单栏 &#8594; 分析 &#8594; 绘制剖面)。 
		注意: 打开一个新窗口将会显示配置文件对应于每个像素所选通道 （具体荧光） 的信号强度为所选的 z 平面节线沿线。
		<ol><li>重复的过程对于每个通道对应使用的荧光 （即， 一个量化为 488，一个用于 568，，一个为 405）。 
			注: 信号强度值将总是订购后绘制的线条的方向。</li>
		</ol></li> <li>获得的数值对应于追求量化的选定 z 平面层的配置文件中每个像素灰度值，复制配置文件数据 (在图像窗口中的菜单栏 &#8594; 副本) 并将其粘贴在任何电子表格软件。</li>
		重复步骤 <li>4.1.1-4.1.4 获取的共聚焦图像的每个 z 平面部分和每个通道在哪里需要量化。 
		注: 可以汇集不同 z 平面层的量化，通过计算每个 z 平面层线沿线的每个像素的总和价值获得特别富集的信号的一般评估。如果绘制的线条是平均的值中包含每个 z 平面层相同，只可以实现这种类型的池。</li>
		<li>作为全细胞定量化不同的渠道，与多层数的一种替代方法，使用的宏，称为 StackprofileData （见 补充文件）。 
		注: 此宏是可以自由地在 https://imagej.nih.gov/ij/macros/StackProfileData.txt，可以使用如下: <ol><li>粘贴到一个文本文件中的宏并保存它。</li>
			<li>打开的图像文件，包括所有细胞的共焦层。</li>
			<li>画一条线，如步骤 4.1.2 所述，打开一个新的窗口，要导入的宏 (菜单栏 &#8594; 插件 &#8594; 宏 &#8594; 运行)，选择对应的 StackprofileData 宏，以前保存的文本文件，然后单击 &#34; 打开 &#34; 
			注: 以下宏输入一个新的窗口命名 &#34; 结果 &#34; 将打开，所有的量化确定轴将列出每个 z 平面层和通道中的图像文件。</li>
			<li>将为每个通道的数据复制 (菜单栏 &#8594; 编辑 &#8594; 副本) 来得到对应信号量化图形化的形象代表。将其粘贴到任何电子表格软件。计算每个 z 平面层线沿线的每个像素的每个通道的总和值。使用这些值来创建图形表示一个与 图 3 给出了相似。</li>
		</ol></li></ol></li> <li>量化信号在指定的细胞面积或体积。
	<ol><li>用 ImageJ 软件打开图像文件。</li>
		<li>绘制面积来表示使用几何形状函数的感兴趣的领域 (工具条 &#8594; &#34; 矩形，&#34; &#34; 椭圆形，&#34; 或 &#34; 多边形 &#34;)。 
		注: 量化值将确定为对应于绘制窗体区域。</li>
		<li>调整要避免任何背景信号的阈值级别 (菜单栏 &#8594; 图像调整 &#8594; 阈值); 一个新的窗口将显示表示像素亮度直方图形式。更改要包括排除使用滑块进行量化处理像素的值。选择一个阈值，消除了红色像素以外的细胞，如以红色突出显示的像素将列入量化。</li>
		<li>定义量化像素信号内所选的区域，没有背景信号的测量参数 (菜单栏 &#8594; 分析 &#8594; 设置测量)，并请务必检查 &#34; 阈值限制 &#34; 和 &#34; 集成密度&#34; 框。</li>
		<li>测量所选区域的定量值 (菜单栏 &#8594; 分析 &#8594; 措施)。 
		注意: 信号强度量化将显示在一个新的窗口下 &#34; IntDen &#34; 鉴定，表示产品的平均灰度值和所选区域内的像素数目。</li>
		<li>绘制包括使用一个几何形式的整个单元格区域 (工具条 &#8594; &#34; 矩形，&#34; &#34; 椭圆形，&#34; 或 &#34; 多边形 &#34;) 并着手步骤 4.2.3-4.2.5 以获得与总信号对应的值。计算所选区域内信号的比值对整个信号获取感兴趣的领域内信号 %。</li>
		<li>重复步骤 4.2.2-4.2.6 以获得每个 z 平面的细胞体积的量化。根据需要进行调整的几何形式。</li>
		<li>复制/粘贴数据源自 &#34; 结果 &#34; windows 为每个 z 平面层到电子表格，图形化的描述，找到均值。</li>
	</ol>
	</li>
</ol>

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J., Rossoll, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+muscular+atrophy:+the+role+of+SMN+in+axonal+mRNA+regulation.">Spinal muscular atrophy: the role of SMN in axonal mRNA regulation.</a> Brain Res. 1462, 81-92 (2012).</li><li>Costa-Mattioli, M., 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=Translational+control+of+hippocampal+synaptic+plasticity+and+memory+by+the+eIF2alpha+kinase+GCN2.">Translational control of hippocampal synaptic plasticity and memory by the eIF2alpha kinase GCN2.</a> Nature. 436, 1166-1173 (2005).</li><li>Costa-Mattioli, M., Sossin, W. S., Klann, E., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+of+long-lasting+synaptic+plasticity+and+memory.">Translational control of long-lasting synaptic plasticity and memory.</a> Neuron. 61, 10-26 (2009).</li><li>Du, T. G., Schmid, M., Jansen, 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=Why+cells+move+messages:+the+biological+functions+of+mRNA+localization.">Why cells move messages: the biological functions of mRNA localization.</a> Semin Cell Dev Biol. 18, 171-177 (2007).</li><li>Ephrussi, A., St Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing:+the+bicoid+morphogen+gradient+matures.">Seeing is believing: the bicoid morphogen gradient matures.</a> Cell. 116, 143-152 (2004).</li><li>Mardakheh, F. K., 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=Global+Analysis+of+mRNA,+Translation,+and+Protein+Localization:+Local+Translation+Is+a+Key+Regulator+of+Cell+Protrusions.">Global Analysis of mRNA, Translation, and Protein Localization: Local Translation Is a Key Regulator of Cell Protrusions.</a> Dev Cell. 35, 344-357 (2015).</li><li>Lacsina, J. R., 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=Premature+translational+termination+products+are+rapidly+degraded+substrates+for+MHC+class+I+presentation.">Premature translational termination products are rapidly degraded substrates for MHC class I presentation.</a> PLoS One. 7, e51968 (2012).</li><li>Schneider-Poetsch, T., 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+eukaryotic+translation+elongation+by+cycloheximide+and+lactimidomycin.">Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin.</a> Nat Chem Biol. 6, 209-217 (2010).</li><li>Jacobs, J. P., Jones, C. M., Baille, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+a+human+diploid+cell+designated+MRC-5.">Characteristics of a human diploid cell designated MRC-5.</a> Nature. 227, 168-170 (1970).</li><li>Gandin, V., 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=Polysome+fractionation+and+analysis+of+mammalian+translatomes+on+a+genome-wide+scale.">Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale.</a> J Vis Exp. (87), (2014).</li></ol>

作者没有透露。

最近的技术进步有允许参与平移上调机制更好地理解或对特定基因的生物学过程，如神经元的发育、 药物反应和转移的镇压。这里所述的符合成本效益方法允许翻译事件在细胞研究 RNA 结合蛋白是如何调节等细胞黏附、 迁移和侵袭转移过程可视化。
虽然已发展了许多方法来评估de novo蛋白质翻译，他们都共享相同的策略，拉长多肽包含标记与检出的基团修饰的氨基酸。...

翻译由不同的细胞功能的调节，促使许多的研究团队，开发新的工具和方法来确定亚细胞定位的 mRNA 翻译和调节的蛋白合成1，2 ，34。这些最近的技术进步允许更好地了解涉及翻译上调或特定基因生物学过程，如神经元的发育、 药物反应和转移5 的镇压的机制 ，，67，8。然而，这些方法大多数需要昂贵或有害试剂和可能不到大多数实验室可用的特定设备。因此，一个具有成本效益的方法，允许翻译事件快速评定了专门绕过这些潜在的问题。此方法检测特定细胞过程中发生的急性平移调制，并且还允许本地化的翻译用共聚焦显微镜。
这里描述的方法被用于监视在亚细胞隔离舱称为传播启蒙中心 (SIC)5的本地化的翻译。结果是在本地化在新生粘附复合物的种子细胞中发现的瞬态结构。虽然结果与粘附复合物是不同的他们的命运被息息相关。的确，结果也会逐渐消失后粘着复杂成熟粘附网站粘附的初始阶段。我们发现已知的具体控制 （例如， Sam68，FMRP 和 G3BP1） mRNA 翻译的 RNA 结合蛋白和 Rna 被内这些丰富的 polyadenylated 结构5。我们使用此处所述的方法，表明 SIC 相关的 mRNA 翻译作为检查点允许调节种子细胞巩固细胞粘附。这种基于嘌呤霉素纳入的方法可以考虑翻译测定 （日落） 的表面传改编的版本。最初开发用来衡量全球蛋白质合成率使用非放射性标记的氨基酸，蛋白质 puromycilation 提供可视化de novo蛋白质合成9的有效途径。这种方法依赖于嘌呤霉素，阻止通过过早链终止在核糖体10翻译抗生素的内在行为。事实上，嘌呤是结构上类似于 tyrosyl-tRNA，允许将纳入拉长肽链通过肽键的形成。但是，嘌呤霉素绑定到肽链阻止新的肽键形成与下一个氨酰-tRNA，嘌呤霉素以来非水解酰胺键而不是在转运中找到的水解酯键。因此，嘌呤霉素纳入拉长多肽产生过早披露的众多截断的 puromycilated 多肽对应积极翻译 mRNA9，11，12.
使用此方法，有可能评估活动翻译内短时间寡妇 (例如， 5 分钟) 期间使用针对嘌呤霉素对 5 分钟时间，辅以抗生素的细胞特异性抗体的细胞粘附在不同的时间点在细胞粘附过程5。这种测定方法的精度依赖于针对 puromycilated 基团的高度特异性抗体。Puromycilated 多肽免疫荧光法检测提供新翻译的 mRNA，也可以使用共焦显微成像系统的精确地量化一般亚细胞重新分区。
因此，此方法提供了大量的实验室研究平移监管机制所涉及的过程，如神经元造粒6，，1314，相关选项15、 形态 mRNA 本地化和翻译期间发展16，17。它也非常适合于快速生物事件，如细胞迁移、 粘连或入侵，或简单地评估可能诱发平移变化5，的药物治疗期间的本地化或条块分割的翻译研究7,18.总体而言，此方法允许为本地化或控制翻译事件的可视化快速、 精确，并且具有成本效益的方式。

<table><tbody><tr><td>DMEM</td><td>wisent</td><td>319-005-CL</td><td></td></tr><tr><td>Trypsine</td><td>wisent</td><td>325-043 EL</td><td></td></tr><tr><td>FBS</td><td>Thermo Fisher Scientific</td><td>12483020</td><td></td></tr><tr><td>Puroycin antibody 12D10</td><td>EMD millipore</td><td>MABE343</td><td>western blot dilution 1:25,000&lt;br/&gt; Immunofluoresence dilution 1:10,000</td></tr><tr><td>Anti-mouse IgG, HRP-linked Antibody</td><td>cell signaling technology</td><td>7076</td><td>western blot dilution 1:8,000</td></tr><tr><td>Western Lightning Plus-ECL</td><td>Perkin Elmer</td><td>NEL104001EA</td><td></td></tr><tr><td>Anti-mouse IgG (H+L), F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment (Alexa Fluor 488 Conjugate)</td><td>cell signaling technology</td><td>4408</td><td>immunofluoresecence dilution 1:400</td></tr><tr><td>CF568 Phalloidin</td><td>biotium</td><td>00044</td><td>immunofluoresecence dilution 1:400</td></tr><tr><td>Cyclohexmide</td><td>Sigma</td><td>C1988-1G</td><td>50&amp;micro;g/ml final concentration</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Invitrogen</td><td>D1306</td><td>final concentration 1&amp;micro;g/ml</td></tr><tr><td>Puromycin</td><td>bio-Basic</td><td>PJ593</td><td>2.5&amp;micro;g/ml to 10&amp;micro;g/ml</td></tr><tr><td>Ibidi &amp;micro;-Dish 35 mm, high, ibiTreat</td><td>Ibidi</td><td>81156</td><td></td></tr><tr><td>MRC-5 cells</td><td>ATCC</td><td>CCL-171</td><td></td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>CCL-2</td><td></td></tr><tr><td>Huh-7 cells</td><td>from Dr. Mazroui (Universit&amp;eacute; Laval)</td><td></td><td></td></tr><tr><td>Fv1000</td><td>olympus</td><td></td><td>confocal imaging system</td></tr><tr><td>Fiji software</td><td></td><td></td><td>http://fiji.sc</td></tr><tr><td>PBS (Phosphate BuffeRed Saline)</td><td>bio-Basic</td><td>PD8117</td><td></td></tr><tr><td>Formaldehyde 37% Solution</td><td>bio-Basic</td><td>C5300-1</td><td></td></tr><tr><td>Triton X-100</td><td>bio-Basic</td><td>TB0198</td><td></td></tr><tr><td>BSA</td><td>Fisher Bioreagents</td><td>BP9702-100</td><td></td></tr><tr><td>Tween20</td><td>Fisher Bioreagents</td><td>BP337-500</td><td></td></tr></tbody></table>

quantitative immunofluorescence to measure global localized translation

调节 mRNA 翻译机制被参与不同的生物学过程，如胚线发育、 细胞分化和器官，以及多种疾病。众多的出版物有令人信服地表明具体机制严格管制 mRNA 翻译。兴趣翻译诱导调控蛋白表达的增加导致了发展的研究并遵循de novo蛋白质合成中 cellulo的新方法。然而，这些方法大多数都复杂，使其成本高昂和经常限制的数量可以进行研究的 mRNA 指标。这份手稿提出要求只基本试剂和荧光共焦成像系统测量并可视化 mRNA 翻译中任何细胞株在不同条件下发生的变化的方法。此方法是最近用来显示本地化的翻译中贴壁细胞的亚细胞结构在短短的时间，从而提供了可视化de novo翻译短期间各种生物的可能性过程或更改进行验证，在翻译活动中对特定刺激的反应。

要准确地观察使用嘌呤霉素纳入翻译事件，它关键是确定每个单元格的行的最佳条件，因为每个显示11，不同嘌呤霉素纳入动力学 (图 1)9 ，，1218。因此，若要验证嘌呤霉素纳入，有必要治疗所需单元格行与标准谱的嘌呤浓度增加 （1.0、 2.5、 5.0、 7.5、 1...

Watch this Scientific Journal Video about Quantitative Immunofluorescence to Measure Global Localized Translation at JoVE.com

Quantitative Immunofluorescence to Measure Global Localized Translation

这份手稿描述了一种方法来可视化和量化本地化的翻译事件亚细胞的车厢内。提出这份手稿的做法需要基本的共焦成像系统和试剂，并且是快速和具有成本效益。

定量荧光测量全球本地化翻译

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

quantitative-immunofluorescence-to-measure-global-localized

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Research

JoVE Journal

Biochemistry

9.8K 次观看.  Université Laval. 这份手稿描述了一种方法来可视化和量化本地化的翻译事件亚细胞的车厢内。提出这份手稿的做法需要基本的共焦成像系统和试剂，并且是快速和具有成本效益。

视频: Quantitative Immunofluorescence to Measure Global Localized Translation

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

遗传学

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

生物化学

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

免疫与感染

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

生物学

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

免疫荧光定量小鼠脑突触蛋白的异种分布

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

神经科学

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

工作流的高含量，荧光标记的万能工具显微镜数据，支持开源软件的单个细胞定量

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

高尔基蛋白质荧光质量中心的定量定位

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.

We describe the use of quantitative immunoblotting to validate immunofluorescence histology coupled with image analysis as a means of quantifying a protein of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Our results demonstrate the utility of immunofluorescence histology for ascertaining the relative quantity of biomarker proteins in routine biopsy samples.

细胞系定量免疫印迹作为常规组织样本中生物标志物蛋白定量免疫荧光定量的标准

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An ...

定量荧光测量全球本地化翻译

摘要

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此视频中的章节

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

用于分析依赖帽的翻译动力学的体外单分子成像测定

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

在哺乳动物细胞中的内质网定位的mRNA的可视化

免疫荧光定量小鼠脑突触蛋白的异种分布

工作流的高含量，荧光标记的万能工具显微镜数据，支持开源软件的单个细胞定量

定量免疫检测，以衡量在中心体的变异蛋白水平

高尔基蛋白质荧光质量中心的定量定位

细胞系定量免疫印迹作为常规组织样本中生物标志物蛋白定量免疫荧光定量的标准

定量荧光测量全球本地化翻译

摘要

探索更多视频

此视频中的章节

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

在哺乳动物细胞中的内质网定位的mRNA的可视化

免疫荧光定量小鼠脑突触蛋白的异种分布

工作流的高含量，荧光标记的万能工具显微镜数据，支持开源软件的单个细胞定量

定量免疫检测，以衡量在中心体的变异蛋白水平

高尔基蛋白质荧光质量中心的定量定位

细胞系定量免疫印迹作为常规组织样本中生物标志物蛋白定量免疫荧光定量的标准

用于分析依赖帽的翻译动力学的体外单分子成像测定