作者感谢穆罕默德 Navab, 艾伦 Fogelman 和 Srinivasa 的工作, 他们在开发这个模型的早期迭代的关键作用。T.A.A. 得到 RMIT 大学校长博士后奖学金的支持。AJ 和 AH 得到了澳洲项目赠款1108792的支持。传统知识得到 nih 资助 nih K08AI08272、nih/NCATS 补助金µL1TR000124。

所有使用人体生物样本的实验都是在加州大学洛杉矶分校和墨尔本阿尔弗雷德医院人类伦理委员会的道德认可下进行的. 
 注意: 荧光 HDL 函数检测有许多变体 (请参见讨论) 32 。下面我们将描述给出最一致和可重现的结果的协议。检测的概述见 图 2 . 
 1. 标本处理 <ol> <li> 使用新鲜、non-hemolyzed 的血清 (可在血清分离管中收集) 或在禁食12-14 小时后获得的枸橼酸血浆。如果使用冷冻保存的样品, 请包括本协议中所述的适当控制. </li>
</ol> 2。0天-化验准备 <ol> <li> 使用适当的控件、示例和复制准备实验的工作布局。运行每个样本至少在 triplicates。有代表性的96井布局显示在 图 3 中. </li>
	<li> 根据样本数和复制量计算将用于化验的所有试剂体积. 
	注: 在每个工作库存中增加10% 的体积, 以确保每个试剂有足够的体积用于特定的实验. </li>
	<li> 将所有所需的管子贴在化验的所有不同步骤上, 例如 hdl 的分离, 高密度脂蛋白 C 的测量 (可选) 和 fluorochromeassay. </li>
</ol> 3。1天-准备控件 <ol> <li> 准备研究特定的汇集控件 <ol> <li> 从所有研究样本的汇集血浆或血清中创建一个控制样本。如果在每一个板块运行20样品三份, 结合10和 #181; l 从每个样本到一个200和 #181; 我汇集控制。给临床实验室单独的分, 以确定这种控制的高密度脂蛋白 C 值. 
		注: 使用此汇集控制来标准化化验, 并说明所有可能已知的 (如基质血清与血浆的类型、冻融循环、超低温保存) 和未知的混杂可能影响实验的变异性 27 , 28 . </li>
	</ol> </li> <li> 准备实验室特定的质量控制 (卡塔尔) <ol> <li> 在每个实验室中准备大量的高密度脂蛋白 (例如, 从10健康捐赠者的5毫升血浆中分离的高密度脂蛋白) 和冷冻的几个等分库存以最小化冻融循环. </li>
		<li> 使用至少10不同于此股票的复制来确定 HDLox 的平均值。每个测试中包括的每个 QC 样品的 HDLox 值的可接受范围是在和 #60 内; 15% 此平均值的变化系数. </li>
		<li> 将此集中控制的单独分到临床实验室, 以确定此控制的高密度脂蛋白 C 值 (如40毫克/dL). 
		注: 关于如何使用血库血液创建这些控件的详细方法以前已被描述为 27 、 28 、 32 。根据需要使用额外的卡塔尔 (例如一个健康的捐赠者已知有正常的 hdl 功能, 一个来自已知有很大异常的 hdl 功能的捐献者. </li>
	</ol> </li> <li> 优化背景信号和空白值 (可选) <ol> <li> 以最小化背景, 在孵化介质中添加适量的过氧化氢酶 (1-4 U/毫升), 以快速去除形成的 H 2 O 2 由于缓冲的自发空气氧化作用. 
		注: 由于从高密度脂蛋白样本的值中减去了从空白井 (没有高密度脂蛋白) 的值, 并且报告的结果相对于一个汇集控制 (包括在同一个96井板中), 我们发现这一步并没有实际 affec结果。因此, 如果需要, 可以省略用过氧化氢酶优化背景信号. </li>
	</ol> </li> </ol> 4。1天-使用高密度脂蛋白沉淀分离高密度脂蛋白胆固醇 注意: 根据制造商和 #39, 使用商业上可用的标准化高密度脂蛋白胆固醇沉淀试剂分离 apoB 衰竭血清;说明.这些试剂广泛用于色度测定, 以确定高密度脂蛋白胆固醇水平. 
<ol> <li> 使用新鲜 (或解冻) 等离子或血清样本. </li>
	<li> 将等量的容积 (如80和 #181; L) 与血浆和高密度脂蛋白胆固醇沉淀试剂 (20% 的聚乙二醇在甘氨酸缓冲液中 pH 10.0 (25 和 #176; C)). </li>
	<li> 上下移混合. </li>
	<li> 1000-2000 x g 离心10分钟. </li>
	<li> 吸气上清液 (HDL 分数). </li>
	<li> 理想情况下, 立即使用隔离的高密度脂蛋白 fluorochromeassay 脂质过氧化。然而, 如果在同一天运行的几个样本和其他步骤, 如测量高密度脂蛋白胆固醇浓度也做了, 然后存储在4和 #176 的隔离高密度脂蛋白, 并在第二天使用它的 fluorochromeHDL 功能测定. </li>
</ol> 5。1天-在分离的高密度脂蛋白中测定高密度脂蛋白-c 注意: 如果从临床实验室中使用高密度脂蛋白 c 的值, 用 hdl-c 的量来规范化 HDLox, 这是可选的. 
<ol> <li> 使用标准色度检测 27 从等离子体中量化高密度脂蛋白胆固醇。在每个井中添加50和 #181 的胆固醇试剂, 并使用比色板读卡器和每个试剂盒中提供的胆固醇标准确定胆固醇浓度. </li>
</ol> 6。2天-试剂的制备 <ol> <li> 制定 hrp 5 u/ml 解决方案的 hrp 和胆固醇溶液 (范围 1-10 u/毫升) </li>
	<li> 准备20毫米荧光 (如 Amplex 红色) 解决方案: 将一小瓶荧光试剂和亚甲基亚砜解冻到室温。在使用前, 在200和 #181 中溶解荧光试剂 (1 毫克); 亚砜 L。存储库存解决方案冻结在和 #8804;-20 和 #176; C, 保护免受光. </li>
	<li> 准备正负控制: 使用1X 和 #39; 反应缓冲器和 #39; 无胆固醇和20毫米 H 2 O 2 工作解决方案分别为负和正控制. </li>
</ol> 7。2天-荧光检测 <ol> <li> 添加50和 #181; L 1x 反应缓冲器为空白 (负控制). </li>
	<li> 可选: 添加 20 mM 工作解决方案 H 2 O 2 作为每个板块的正向控制. </li>
	<li> 为了尽量减少实验的变异性, 并确保添加试剂和样品, 并及时地进行, 在一个单独的96井圆底, 透明, 聚苯乙烯或聚丙烯板 (板 1) 的所有添加样品。然后使用多通道吸管将特定的体积转移到 3 96 井板 (板 2-4: 聚丙烯, 扁平机器人汤姆, 黑色) (具有相同的布局) ( 图 2 , 图 3 )。
	<ol> <li> 例如, 首先添加160和 #181; 独立的高密度脂蛋白 l 的每个井/样本 1, 然后与多通道吸管转移50和 #181; l 的高密度脂蛋白胆固醇从每个井/样本进入96井黑板。更改各行/列之间的提示, 并使用筛选提示进行所有转移. </li>
	</ol> </li> <li> 将样本留在井中。不要丢弃。在添加试剂之间没有洗涤步骤. </li>
	<li> 添加50和 #181; HRP 解决方案的 L 5 u/毫升 (0.25 u) 每井. 
	注: 在添加荧光试剂前使用 HRP. </li>
	<li> 在37和 #176 孵育30分钟;在孵化后不要丢弃样品 (在添加试剂之间没有洗涤步骤). </li>
	<li> 添加50和 #181; fluorochromereagent 为300和 #181 的最终浓度; m。在这一点上, 总反应量是150和 #181; l 混合良好和保护从光. </li>
	<li> 评估荧光读数 (在黑暗中) 每分钟超过120分钟, 在37和 #176; C 与荧光板阅读器 (530/590 nm 过滤器). 
	注: 使用较短的60分钟间隔与新鲜样品。我们发现, 120 分钟的化验提供更多的可重现性的数据与使用冷冻保存的样品. </li>
	<li> 使用适当的软件记录数据. </li>
</ol> 8。3天-数据分析 <ol> <li> 记录荧光单位 (任意单位), 在120分钟后添加荧光试剂, 包括空白井和所有控制. </li>
	<li> 计算荧光单位的平均值 (基于至少 triplicates) (HDL ox_sample )。不要把离群值 (和 #62, 2 SDs 从平均值) 考虑进去. </li>
	<li> 使用下列公式减去背景荧光: 
	<img alt="Equation 1" src="/files/ftp_upload/56325/56325eq1.jpg"/> </li> <li> hdl 胆固醇量的规范化 (高密度脂蛋白 c): 对每个样本 (包括汇集的控制) 的 HDLox 脂质过氧化值正常化 (毫克/dl)。在整个结果部分, hdl 牛 被显示为 n (hdl-c) HDLox 测量, 以反映 hdl-c 的调整。使用以下公式: <img alt="Equation 2" src="/files/ftp_upload/56325/56325eq2.jpg"/> </li> <li> 将每个样本的 HDLox 脂质过氧化值的表达式标准化, 使其与池控件的值对。正常化的 n (hdl-c) HDLox 脂质过氧化值为每个样本 (调整为高密度脂蛋白 c) 的 n (hdl-c) HDLox 脂质过氧化值的汇集控制。在整个结果部分, hdl 牛 被提出作为 nHDL 的 牛 措施, 以反映实验变异性和高密度脂蛋白 C 的调整。使用以下公式: <img alt="Equation 3" src="/files/ftp_upload/56325/56325eq3.jpg"/> 注意: 此方法类似于其他已建立的实验方法, 以减少测量的实验可变性, 如国际正常化比率 (INR) 44 , 45 。此方法已在临床研究中得到验证 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 . </li> <li> 使用标准质量控制示例对实验结果进行质量控制。每个实验室都应该建立自己的质量控制 (卡塔尔)。理想情况下, 有一个 qc 为 nHDLox 从一个已知功能失调的高密度脂蛋白样本和 qc nHDLox 从一个样本从健康的捐助者 [例如使用汇集样本从年轻的成人谁是建立血库捐助者, 并没有已知的共病和危险因素包括吸烟在内的心血管疾病)。后者控制标准化结果在不同的实验室之中。这些卡塔尔的平均值是基于至少5复制而建立的 (通常我们对这个重要步骤使用10复制)。
	<ol> <li> 检查每个测试中测量的卡塔尔是否在预期的值范围内有值。假设一个可接受的最大实验变异率为 15%, 所有测量的实验值应该在已知的标准质量值的15% 以内。使用下列公式: <img alt="Equation 4" src="/files/ftp_upload/56325/56325eq4.jpg"/> <img alt="Equation 5" src="/files/ftp_upload/56325/56325eq5.jpg"/> 注释: 如果其中一个包含的质量控制示例 (正常与机能失调的高密度脂蛋白) 给出的 HDLox 值超出预期范围 (在每个实验室建立) 然后重复实验. </li>
	</ol> </li> <li> 使用变体系数 (CV) 对实验结果进行质量控制, 以量化实验可变性: 用 CV% 和 #62 重复所有样品; 15%。使用以下公式: <img alt="Equation 6" src="/files/ftp_upload/56325/56325eq6.jpg"/> 注意: 一个典型的 intra-assay (在同一个板块内) 和中间 (在不同的板块之间) 实验变异是和 #60; 15%. 2) 一个典型的intra-assay cv 介于1-7% 和一个典型的 interassay cv 之间 3-10%. </li>
</ol>

本协议和化验与专利 PCT/US2015/018147 有关。

这里描述的协议提供了一个强有力的工具来回答关于高密度脂蛋白功能在动脉粥样硬化和人类疾病中的作用的重要研究问题。用酶促扩法 (HRP) 对 hdl-C 的每毫克高密度脂蛋白脂质过氧化物含量进行定量分析。这种方法可以避免已知的 HDL 功能检测的局限性 (如胆固醇外流测定), 包括对所用细胞类型的显著异质性, 报告的读数类型, 缺乏标准化和混杂效应甘油三酯7,<sup class="xr...

动脉粥样硬化性心血管疾病 (CVD) 是导致全球死亡的主要原因1,2。流行病学研究表明, 低浓度高密度脂蛋白 (HDL) 胆固醇通常与动脉粥样硬化发展的风险成反比1,2。虽然几项研究支持高密度脂蛋白1,2的 atheroprotective 作用, hdl 减轻动脉粥样硬化的起始和进展的机制是复杂的3,4。因此, 有人建议, HDL 的复杂结构和功能, 而不是绝对水平可以更准确地预测动脉粥样硬化5,6,7,8。一些高密度脂蛋白蛋白和脂质成分的改变会损害 hdl 的功能, 如动脉粥样硬化等炎症状态。这些 i) 降低其胆固醇流出电位9, ii) 减少消炎和增加 HDL 相关的炎蛋白6,7, iii) 降低抗氧化因子水平和活性和 HDLs抑制低密度脂蛋白氧化的能力 (LDLox)10和 iv) 增加脂质过氧化氢含量和氧化还原活性 (HDLox)9,11。对 hdl (如胆固醇外流、抗氧化功能) 的 pleotropic 功能进行评估的健壮性分析可以补充临床上 hdl-高密度脂蛋白 C 的测定。
HDL 功能通常由细胞方法评估, 如胆固醇流出试验8,12,13,14。这些方法有很大的局限性, 包括对所使用的细胞类型、报告的读数类型、缺乏标准化和甘油三酯的混杂效应7,15。这些缺点给大型临床研究带来困难16。与细胞检测相比, 无单元检测可能给 HDL 功能提供更有力的措施。胆固醇外流是 HDL 最重要的功能之一, 但它只能通过细胞检测来确定。其他方法来确定 HDL 功能, 如蛋白质组学17,18,19,20,21,22,23, 24和细胞单核蛋白的趋化性分析 HDL 功能17,22,25尚未标准化, 不能用于大规模的人类研究。
HDL 具有显著的抗氧化剂 atheroprotective 效果5,6,7,8。HDL 的抗氧化功能已确定在 LDL 的存在前细胞游离荧光分析26。这些生化荧光方法的高密度脂蛋白抗氧化功能最初开发的穆罕默德 Navab 和艾伦 Fogelman 及其同事26。虽然许多人类研究使用这些方法来确定 HDL 功能 17,18,19,20,21,22,23 ,24, 脂质 (高密度脂蛋白) 脂质 (LDL) 和脂质荧光相互作用可能会限制这些细胞游离酶生化检测 HDL 功能的重复性27,28。
最近的兴趣集中在高密度脂蛋白氧化的功能后果, 这是脂质和蛋白在 hdl 27,29,30中的氧化作用的结果。先前的研究表明, hdl 的氧化作用会损害 hdl 的功能27,29,30。高密度脂蛋白在脂质过氧化物转运中有重要作用, 高脂质过氧化物与 hdl 功能异常有关。因此高密度脂蛋白脂质过氧化物含量可用于确定 hdl 功能9,17,20,31 , 并给出已知的 hdl 函数前分析的局限性7, 15、27、32, 我们开发了一种可量化 HDL 脂质过氧化物含量 (HDLox) 32的替代荧光方法。这种方法是基于酶辣根酶 (HRP) 和荧光 Amplex 红, 可以量化 (不含胆固醇氧化酶) 的脂质过氧化物含量每毫克的 HDL-C 32。该检测的生物化学原理显示在图 1中。我们已经表明, 这种荧光方法没有先前的 HDL 函数分析的限制27,28。本试验在实验室中得到了进一步的完善和规范, 使其能够可靠地用于大规模人类研究, 即使是低温保存的等离子体32,33,34,35,36,37,38,39,40,41,42. 该方法的读数与经验证的细胞检测的读数、心血管疾病的替代措施、全身炎症、免疫功能障碍以及相关心血管和代谢风险表型有关。32,33,34,35,36,37,38,39. 在这里, 我们描述这个简单, 但稳健的方法来测量 HDL 脂质过氧化物含量 (HDLox)。这种方法可以作为一个工具, 回答重要的研究问题的作用, HDL 功能在人类疾病中的系统性炎症, 氧化应激和氧化脂有一个关键的作用 (如动脉粥样硬化)32。

<table>
	<tbody>
		<tr>
			<td>Experimental Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HDL PEG (Polyethylene Glycol) Precipitating Reagent</td>
			<td>Pointe Scientific</td>
			<td>H7511</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex Red reagent.</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>DMSO.</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>Horse Radish Peroxidase (HRP)</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>Cholesterol Esterase.</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>&#160;Cholesterol Reference standard</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>&#160;Resorufin fluorescense Reference standard</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>5x Reaction Buffer.</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>HDL Cholesterol Automated Reagent</td>
			<td>ThermoFisher Scientific Co., San Jose, CA, USA.</td>
			<td>TR39601</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasticware&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates (polypropylene, flat bottom, clear).</td>
			<td>Sigma Aldrich</td>
			<td>M0687</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates (polypropylene, flat bottom, black).</td>
			<td>Sigma Aldrich</td>
			<td>M9936</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>0030 125.150</td>
			<td></td>
		</tr>
		<tr>
			<td>ClipTip 200, sterile</td>
			<td>ThermoFisher Scientific Co., San Jose, CA, USA.</td>
			<td>14-488-058</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Multichannel Pipettes, 8-channel, 125&#160;</td>
			<td>ThermoFisher Scientific Co., San Jose, CA, USA.</td>
			<td>&#160;14-387--955</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5 2.01 software</td>
			<td>Biotek, Vermont, USA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5 Plate reader</td>
			<td>Biotek, Vermont, USA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-free biochemical fluorometric enzymatic assay for high-throughput measurement of lipid peroxidation in high density lipoprotein

低高密度脂蛋白胆固醇 (HDL C) 水平是动脉粥样硬化性心血管疾病 (CVD) 最强大的独立阴性预测因子之一。hdl 的结构和功能, 而不是高密度脂蛋白 C 可以更准确地预测动脉粥样硬化。一些高密度脂蛋白蛋白和脂质成分的改变会损害 hdl 的功能, 如动脉粥样硬化等炎症状态。HDL 的功能通常是由细胞基础的化验, 如胆固醇外流化验, 但这些化验有许多缺点缺乏标准化。与细胞检测相比, 无单元检测可能给 HDL 功能提供更有力的措施。高密度脂蛋白氧化损害 hdl 功能。高密度脂蛋白在脂质过氧化物转运中有重要作用, 高脂质过氧化物与 hdl 功能异常有关。在解释酶荧光法测定脂质氧化状态的结果时, 应考虑脂探针的相互作用。这促使我们开发一种无细胞生物化学酶方法来评估 hdl 脂质过氧化物含量 (HDLox), 从而导致高密度脂蛋白功能障碍。这种方法是基于酶辣根酶 (HRP) 和荧光 Amplex 红, 可以量化 (不含胆固醇氧化酶) 的脂质过氧化物含量每毫克的高密度脂蛋白 C。这里的一个协议是 describedfor 测定 HDL 脂质过氧化荧光试剂。实验条件的严格标准化可以降低测定的变异性。较高的 HDLox 值与降低的 HDL 抗氧化功能相关。该方法的读数与经验证的细胞检测的读数, 心血管疾病的替代措施, 全身炎症, 免疫功能障碍, 以及相关的心血管和代谢风险表型有关。这种技术方法是一种强有力的方法来评估 HDL 功能在人类疾病中, 全身炎症, 氧化应激和氧化脂有一个关键的作用 (如动脉粥样硬化)。

50µL 每个高密度脂蛋白样本添加到每个井和步骤7.3。50µL 的 HRP 溶液 5 u/毫升 (0.25 u), 然后添加到每个井, 在步骤7.5。样品在37° c 时孵育30分钟, 如步骤7.6 所示。50µL 的荧光试剂, 然后添加到每个井, 在步骤 7.7 (最终浓度300µM)。荧光读数 (在黑暗中), 然后评估每分钟超过120分钟, 在37° c 的荧光板阅读器 (530/590 nm 过滤器)。有代表性的荧光数据的空白, 汇集控制, 样品与已知的功?...

Watch this Scientific Journal Video about Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein at JoVE.com

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

我们在这里描述了一种荧光无细胞生化测定高密度脂蛋白脂质过氧化的方法。这种快速和重现性的分析可以用来确定高密度脂蛋白功能的大规模研究, 并能有助于我们的理解 hdl 功能的人类疾病。

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

cell-free-biochemical-fluorometric-enzymatic-assay-for-high

Research

JoVE Journal

Immunology and Infection

9.2K 次观看.  University of California, Los Angeles. 我们在这里描述了一种荧光无细胞生化测定高密度脂蛋白脂质过氧化的方法。这种快速和重现性的分析可以用来确定高密度脂蛋白功能的大规模研究, 并能有助于我们的理解 hdl 功能的人类疾病。

视频: Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

科研

免疫与感染

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

高通量检测到的表型<em&gt;沙门氏菌</em&gt;伤寒协会，侵袭和复制的巨噬细胞

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

胞外DNA释放的高通量测量和人中性粒定量NET形成<em&gt;体外</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

高通量基因定位在<em&gt;布氏锥虫</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

一种高通量、高含量、液态的 Pathosystem线虫

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...