1。系统配置<ol><li>架F-7000荧光光谱仪配备高感光度扩展范围的R-928F探测器的光电倍增管。 </li><li>使用的F-7000附件：罗丹明B，光扩散，红色过滤器和不合标准的光源产生的光谱仪器的校正因子。 </li><li>量子产量测量附件，其中包括：60毫米，积分球，三氧化二铝白色的瓷砖，白色标准Spectralon粉细胞（2EA），氧化铝粉和量子产率的软件。 </li><li>报告生成程序和相应的模板将用于色度坐标计算。 </li></ol> 2。系统设置<ol><li>打开日立F-7000荧光光谱仪，使氙灯预热一小时。开始使用安装在仪器的标准样品室（试管架）。 </li></ol> 3。 ACQuisition的积分球校正因子测量积分球的校正因子时，软件会自动选择表1中列出的参数测量试验。 <table border="1"><tbody><tr><td colspan="2"> 分析条件 </td></tr><tr><td>测量</td><td>波长扫描</td></tr><tr><td>扫描模式</td><td>同步</td></tr><tr><td>数据模式</td><td>荧光</td></tr><tr><td>电磁轮候册</td><td> 200纳米</td></tr><tr><td> EX开始轮候册</td><td> 200纳米</td></tr><tr><td> EX完轮候册</td><td> 900毫微米</td></tr><tr><td>扫描速度</td><td> 240纳米/分钟</td></tr><tr><td>延迟</td><td> 5.0小号</td></tr><tr><td> EX狭缝</td><td> 5.0纳米</td></tr><tr><td>电磁缝</td><td> 20纳米</td></tr><tr><td> PMT电压</td><td> 250伏</td></tr><tr><td>纠正谱</td><td>作者： </td></tr><tr><td>响应</td><td>汽车</td></tr></tbody></table> 表1。 3.1。收购漫射数据<ol><li>放置在标准的样品室的扩散，并关闭样品室。 </li><li>点击窗口上的量子产率修正系数的测量，然后在扩压测量。 </li><li>输入文件名 ​​：“IS_factor_F70_diffuser”扩散数据，并单击“确定”（ 视频1）。 </li><li>经过测量，该文件将被保存在“正确”的佛罗里达州解决方案的文件夹， 图1是一个扩散器测量数据的例子。 </li></ol> 3.2。没有样品采集校正系数（参考） &lt;OL&gt; <li>从仪器上取下，商店扩散标准​​的样品室，然后安装在积分球。 </li><li>充满了高度至少25毫米，以确保粉末充分涵盖了积分球端口的氧化铝粉粉末细胞。点击底部的细胞仔细压缩粉末。 </li><li>参考端口（P2）的积分球（一个面临排放单色）和氧化铝采样口（P1）的积分球（粉细胞放置在白色瓷砖的三氧化二铝面临的激励单色）。 </li><li>量子产率修正系数的测量窗口上点击，然后在积分球测量（不含样品）（ 视频2）。 </li><li>该软件会提​​醒你设置的样本。输入“ 是没有样本 ”的名称，并单击“确定”。 </li><li>经过测量，该文件将被保存在文件夹“校正<html> T“FL的解决方案。 图2是一个没有样品的测量数据的积分球的例子。 </li></ol> 3.3。采集样品存在校正因子： <ol><li>与三氧化二铝粉末的细胞取出，并更换它与Spectralon白色标准。 （Spectralon标准应该是面对激发单色器（P1））。 </li><li>量子产率修正系数的测量窗口上点击，然后在积分球测量（样品）。该软件会提​​醒你设定的标准测量反射材料。 </li><li>输入名称：“与样本 ”积分球与样本数据文件，并单击OK（ 视频3）。 </li><li>经过测量，该文件将被保存在“正确”的佛罗里达州解决方案的文件夹。 图3是一个与测量数据样本的积分球的例子。 </li></ol>“&gt; 注：如果您需要使用截止过滤器来阻止二阶散射光的干扰荧光峰的发射端，您将需要使用的扩散和扩散，并适当的过滤器测量基准这些。数据文件将被使用的软件时，量子产率计算，在实践中，我们建议所有的系统设置的初始部分的截止过滤器测量。 4。样品测​​量（水杨酸钠粉） 量子产率的测量涉及收购都没有样本（参考），并在样品存在的发射光谱。选择分析测量参数如下： <ol><li>点击“方法”按钮，在常规选项卡选择波长扫描测量模式，并输入适当的信息运营商及配件（ 视频4）。 </li><li>点击“工具”选项卡，并输入仪器的测量参数，如表2（视频5）所示。 </li></ol><table border="1"><tbody><tr><td colspan="2"> 分析条件 </td></tr><tr><td>测量</td><td>波长扫描</td></tr><tr><td>扫描模式</td><td>排放</td></tr><tr><td>数据模式</td><td>荧光</td></tr><tr><td> EX轮候册</td><td> 350纳米</td></tr><tr><td>新兴市场开始轮候册</td><td> 330 nm处</td></tr><tr><td>电磁结束轮候册</td><td> 600纳米</td></tr><tr><td>扫描速度</td><td> 1200纳米/分钟</td></tr><tr><td>延迟</td><td> 0秒</td></tr><tr><td> EX狭缝</td><td> 5.0纳米</td></tr><tr><td>电磁缝</td><td> 5.0纳米</td></tr><tr><td> PMT电压</td><td>为350 V </td></tr><tr><td>纠正谱</td><td>作者： </td></tr><tr><td>响应</td><td>汽车</td></tr><tr><td>纠正谱</td><td>作者： </td></tr></tbody></table> 表2。 <ol start="3"><li>不需要额外的设置都需要在这个时候，因为已测数据后，可监控，处理和报告“选项卡设置。我们只是对其进行审查，然后点击“确定”按钮，以设置在仪器（ 视频6）选定的测量参数。 </li><li>作为一个选项，选择设置可以保存供将来使用。我们现在将着手测量氧化铝的参考标准，采用直接激发。 </li><li>将粉细胞样本中测量端口（P1）（在前面的激发光束）与氧化铝粉。 </li><li>点击“示例”按钮键入样品名称：“P1_Baseline_Al2O3”，然后点击“自动文件”旁边的框。选择的数据文件夹和文件的名称：“P1_Baseline_Al2O3”，然后单击“保存”和“确定”（ 视频）。 </li><li> “办法”按钮上点击Al2O3样品的测量。 （ 视频8），数据处理窗口打开后，点击“自动刻度轴”按钮，调整规模，以可视化的直接激发（ 图4）散射峰。 </li><li>现在，我们将继续使用直接激发水杨酸钠测量样品。点击“样品”图标，进入“ 水杨酸P1_Sodium”的样品和文件名 ​​，然后单击OK按钮（ 视频9）。 </li><li>放置在粉细胞，并在积分球（港口面临的激发光束）的P1口水杨酸钠样品，并点击“测量”按钮。 （ 视频10）当数据的过程ING窗口打开后，点击“自动刻度轴”按钮，调整规模和可视化的散射和荧光峰。 </li><li>在这个时候，我们将重复使用的样品放置在积分球的P2口为三氧化二铝和水杨酸钠的测量，为了阅读使用间接激励。 </li><li>首先点击“样本”按钮和类型的样品和文件名​​：“P2_Baseline_Al2O3”（视频11）。 </li><li>将氧化铝白色瓷砖从P2 P1的积分球，放在一个细胞充满P2中铝氧化物粉末。 </li><li> “办法”按钮，点击读取样本（ 视频12）。 </li><li>完成样品的测量，我们需要衡量水杨酸钠样品使用间接照射。首先，我们在前面的步骤中键入样品和文件名。该名称将成为P2_Sodium水杨酸 （ 视频13）。 </li><li>水杨酸钠样品放置在积分球的P2口，然后按一下测量按钮（ 视频14）。 </li></ol> 5。量子产率计算首先，我们将继续装载积分球的校正因子。 <ol><li>量子产率计算按钮，点击打开的量子产率的计算程序（ 视频15）。 </li><li>点击量子产率修正系数的设置按钮（ 视频16）。 </li><li>点击积分球校正选项卡，点击“积分球校正”前的复选框，然后在过滤器校正“选项卡，确保未选中”过滤器校正“框中，单击”再次在积分球更正“选项卡（ 视频17 ）。 </li><li>漫反射测量数据部分点击Load按钮，然后选择“文件”IS_factor_F70样本 “（ <stronG&gt;视频18）。 </li><li>选择“IS_factor_F70_diffuser”的文件，然后点击Load按钮（ 视频19）。 </li><li>点击的积分球测量数据的Load按钮（样本）一节（ 视频20）。 </li><li>选择“IS_factor_F70没有样品 ”文件，然后点击Load按钮（ 视频21）。 </li><li>点击Load按钮的积分球测量数据（样本）一节（ 视频22）。 </li><li>选择“ 样IS_factor_F70”的文件，然后负载按钮（ 视频23）。 </li><li>归波长可以留在600nm的波长值等于1积分球校正或调整。要做到这一点，确保在“显示量子产率计算窗口”前面的复选框被选中，并单击“确定”按钮的“量子产量因子设置”windoW，这将关闭此窗口（ 视频24）。 </li><li>现在的“量子产量计算”窗口“积分球更正”选项卡上单击调整光标，直到积分球校正读为“1”，使波长（ 视频25）。 </li><li>点击“ 量子产率修正系数的设定 ”，如果需要改变波长归在上一步得到的读数，然后单击“确定”按钮（ 视频26）。 </li></ol>下一步是要加载的基线和样本数据文件<ol start="13"><li>点击“量子产率的计算”选项卡（ 视频27）。 </li><li>加载“数据无样品直接照射（ 文件P1_Baseline_氧化铝 ），点击直接照射（ 视频28）负载按钮和负载”样本数据“（ 文件P1_Sodium水杨酸 ）。 </li><li&gt;“下一步，我们将继续选择”散射“和”荧光“地区（ 视频29）。 </li><li>现在，我们将计算出样品直接照射的量子产率。点击“计算”按钮，并宣读结果（ 视频30）。最后考虑到间接激发样品的量子产率计算，我们需要这些数据。 </li><li>点击文本文件，文件名 ​​“QY型直接照射”（ 视频31）下保存数据。 </li><li>使用数据文件P2_Baseline_Al2O3和P2_Sodium水杨酸 ，我们将计算间接激发的量子产率（32视频 ），现在我们将这个数据保存最终的量子产率计算。 </li><li>点击文本文件和保存的名义下“QY型间接辐射”（33）视频数据的文本文件，现在我们将两个文本文件在Excel中打开方向量子产量数据t和间接激励。最后，我们将计算出样品的量子产率，包括间接激励效果，请使用下列公式计算： </li></ol> Φ=ΦD（1-AD）φI 其中： Φ是更正后的量子产率，考虑到间接激发 ΦD是使 ​​用直接激发内部量子产量。 （内部量子产率，荧光/吸收激发光量的金额。） 广告是直接激发吸收。 （这是样品所吸收的激发光量的比例）。 （吸光度=（AREX - CsEx）/ AREX，其中AREX是激发光量和CsEx是反射光量） φI是使用乐悠内部量子产量矩形励磁 Φ= 0.536 - （1 - 0.848）0.420计算 Φ= 0.47216 6。色度计算<ol><li>随着色度计算准备了一个模板，我们将使用可选软件报表生成。 </li><li>打开数据文件P1_Sodium水杨酸（ 视频34）。 </li><li>单击“属性”按钮，然后在“报告”标签“。在“输出”选择“使用的打印发生器表”，从下拉菜单。在“打印项目”选择模板“FL70Std01_Color-chart.xls”，然后单击“打开”按钮。有没有必要选择的波长范围和间隔，因为这样做自动报告生成器（ 视频35）。 </li><li>下一步是创建报告。点击“报告”标签，将执行宏创建报告和样品名称下保存在“报告”在Excel格式的文件夹（ 视频36）。<html> / LI&gt; <li>这时我们可以打开看到的颜色数据（ 视频37）的报告。 </li></ol> 7。成功的秘诀<ol><li>使用新鲜的样本。 </li><li>要知道，来自不同制造商的材料可以得出不同的结果。 </li><li>点选粉细胞底部压缩样品，并提出了一种用于测量表面均匀。 </li><li>由轻保护样品。他们与光线照射恶化。 </li><li>尝试使用一个更快的扫描速度，以尽量减少样品暴露在光线下。 </li></ol> 8。代表结果 8.1。水杨酸钠是已知的有0.4至0.5的量子产率<img src="/files/ftp_upload/3066/3066fig1.jpg" alt="图1" /> 图1。 <a href="http://jove.com/files/ftp_upload/3066/3066fig1large.jpg" target="_blank">点击这里查看大图</a> 。 iles/ftp_upload/3066/3066fig2.jpg“ALT =”图1“/&gt; 图2。 <a href="http://jove.com/files/ftp_upload/3066/3066fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/3066/3066fig3.jpg" alt="图1" /> 图3。 <a href="http://jove.com/files/ftp_upload/3066/3066fig3large.jpg" target="_blank">点击这里查看大图</a> 。

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantum Yield Measurement of Sodium Salicylate. FL080002, 1 (2008).</li><li>Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+Fluorescence+Spectroscopy.">Principles of Fluorescence Spectroscopy.</a> Science and Business Media, LLC. 60, (2006).</li><li>Horigome, J., Wakui, T., Shirasaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Correction+Method+for+Determination+of+Absolute+Fluorescence+Quantum+Yields+of+Solid+Samples+with+a+conventional+Fluorescence+Spectrophotometer.">A Simple Correction Method for Determination of Absolute Fluorescence Quantum Yields of Solid Samples with a conventional Fluorescence Spectrophotometer.</a> Bunseki Kagako. 58 (6), 553-559 (2009).</li></ol>

答：路易斯·莫雷诺日立高新技术的美国生产在这篇文章中所使用的仪器是受雇于。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>水杨酸钠粉</td><td>瓦科</td><td> 191-03142 </td><td>摩尔。重量160.10 </td></tr></tbody></table>

absolute quantum yield measurement of powder samples

荧光量子产率的测量在寻找新的解决方案的开发，评估，质量控制和照明的研究已成为重要的工具，视听设备，有机EL材料，薄膜，过滤器和生物产业的荧光探针。 量子产率的计算公式为吸收光子的数目比，材料发出的光子的数量。量子产率更高，更好的荧光物质的效率。 对于在本视频的测量，我们将使用日立F-7000荧光光谱仪配备的量子产量测量附件和报告生成程序。所有提供的信息适用于该系统。 粉末样品中的量子产量测量执行以下步骤： <ol><li>生成激励和emiss的仪器校正因子离子单色。这是一个正确的测量量子产量的重要要求。它已在仪器的测量范围提前进行，并不会由于时间的限制，在这个视频显示。 </li><li>积分球校正因子的测量。这一步的目的是要考虑到用于测量积分球反射特性。 </li><li>参考和样品的测量，使用直接激励和间接激励。 </li><li>量子产率的计算使用的直接和间接激励。直接激发样品时，面临着直接的激发光束，这将是正常的测量设置。然而，因为我们使用积分球，从样品的荧光发射的光子的一部分由积分球反射，将重新激发样品，所以我们需要考虑到间接激励。这是ACCOmplished通过测量样品放置在港口面临的发射单色，计算间接量子产率和纠正直接的量子产率计算。 </li><li>纠正量子计算产量。 </li><li>色度坐标计算使用报告生成程序。 </li></ol>日立F-7000量子效率测量系统提供此应用程序的优势，如下： <ul><li>高灵敏度（S / N比800或更好的有效值）。信号是在下列条件下测量：EX波长为350纳米，带通EX和EM为5 nm，响应2秒）水的拉曼光谱，拉曼峰的最大噪声测量。高灵敏度允许测量样品甚至低量子产率。使用这个系统，我们已经测量低至0.1水杨酸的样本和高镁钨样品0.8量子产率。 </li><li>高度精确的测量动态范围的6个订单大小允许双方尖锐的散射峰强度高，以及广泛的荧光峰强度低，在相同条件下进行测量。 </li><li>高测量吞吐量和减少光线照射到样品，由于到高扫描速度可达60,000海里/分钟，自动快门功能。 </li><li>测量量子产量超过宽的波长范围从240至800 nm。 </li><li>精确的量子产率的测量采集仪的光谱响应和整合球体校正因子测量样品的结果。 </li><li>大型专用和易于使用的软件所提供的计算参数的选择。 </li></ul>在这部影片中，我们将测量粉状水杨酸钠，这是众所周知的量子产值的0.4至0.5。

Watch this Scientific Journal Video about Absolute Quantum Yield Measurement of Powder Samples at JoVE.com

Absolute Quantum Yield Measurement of Powder Samples

在这段视频中，我们将演示绝对量子产率和色度坐标直接粉末样品，用日立F-7000量子效率测量系统的测量和计算。

粉末样品的绝对量子产量测量

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

absolute-quantum-yield-measurement-of-powder-samples

Research

JoVE Journal

Biology

27.9K 次观看.  Hitachi High Technologies America. 在这段视频中，我们将演示绝对量子产率和色度坐标直接粉末样品，用日立F-7000量子效率测量系统的测量和计算。

视频: Absolute Quantum Yield Measurement of Powder Samples

Large Volume (20L+) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column. Two 20L samples can be filtered simultaneously using a single pump unit equipped with four rotating heads. Filtration is done in the field on extended trips, or immediately upon return for day trips. It is important to record the amount of water passing through each sterivex filter unit. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use. This procedure will take approximately 5 hours plus an additional hour for clean up.

Large Volume 20L+ Filtration of Coastal Seawater Samples

This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column.

大容量（20L）过滤沿海海水样品

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

科研

生物学

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

定量组织样本中的γH2AX灶

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

高通量测量和环境样品中的有机磷分类

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

人类颗粒酶在哺乳动物细胞中高产量和经济高效表达系统

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

选择反应监测质谱法进行绝对定量蛋白质

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.
A fixed volume (200 &#181;L) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.
Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/&#181;L, for most of the examined miRNAs.
This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

定量实时反转录 PCR 技术对猕猴猴血浆 MicroRNA 水平的绝对量化

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

小鱼肾注射液循环系统中微粒的简易有效管理与可视化

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Methods Article

粉末样品的绝对量子产量测量