作者希望确认生物标志物和免疫测定产品开发团队对该方法的发展的贡献。此外, 我们还要感谢 Vigene 技术, Inc. 在设计数据分析软件包方面的合作。

所有动物实验都是根据 NIH 的建议进行的, 它们包含在实验室动物的护理和使用指南中, 并得到了 IACUC 在 BioLegend 的批准. 
 <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56440/56440fig1.jpg"/> 图 1: 过滤板检测程序摘要. 使用滤板进行化验的协议代表了一个描述关键化验成分和孵化步骤的图示。<a href="//ecsource.jove.com/files/ftp_upload/56440/56440fig1large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
 1. 生物样品制备 注意: 所选的解剖部位和血液收集量由每个单独的调查员自行决定, 不会影响化验结果。但是, 在获得这些样本后, 应按照下面列出的步骤来处理示例. 
<ol> <li> 准备血清样本 <ol> <li> 将所需的血液量收集到首选收集管中, 并允许其凝结至少30分钟. </li>
		<li> 在室温下将样品离心10分钟, 1000 x 克. </li>
		<li> 将血清和检测立即或分到聚丙烯离心管中, 并将样品储存在 #8804;-20 和 #176; C. </li>
	</ol> </li> <li> 血浆样品的制备 <ol> <li> 用乙酸酸 (EDTA) 包膜采血管收集所需的血液量. </li>
		<li> 在室温下离心10分钟 1000 x g, 在30分钟内收集. </li>
		<li> 立即将等离子和化验, 或分到聚丙烯离心管中, 并在 #8804 上储存样品;-20 和 #176; C. </li>
	</ol> </li> <li> 准备组织培养上清样品 <ol> <li> 将样品离心10分钟, 1500 x g 在4和 #176; C 移除细胞和碎片. </li>
		<li> 将上清和化验立即或分到聚丙烯离心管中, 并贮存于 #8804;-20 和 #176; C. </li>
	</ol> </li> <li> 解冻生物样品 <ol> <li> 完全解冻以前冰冻的生物样品, 并在使用前短暂的涡旋。避免超过2的冻结/解冻周期. 
		注: 本协议所使用的样品是通过培养 balb/c/c 小鼠脾 (1 x 10 6 细胞在37和 #176 中获得的; c + 5% CO 2 在各种情况下: 刺激, LPS (100 ng/毫升), 和 #945; CD3 (1 和 #181; g/毫升镀膜) + 和 #945; CD28 (1#181; 可溶性), PMA (20 ng/毫升) + Ionomycin (500 ng/毫升)。养殖清在 48 h 之后收集, 并按1.3 节所述处理. </li>
	</ol> </li> </ol> 2。试剂制备 <ol> <li> 预混合抗体固定化珠的制备 <ol> <li> 几种预珠瓶在室温下 sonicator 浴中1分钟, 然后在三十年代使用前涡旋。如果没有可用的 sonicator 浴缸, 则将涡流时间增加到1分钟. </li>
	</ol> </li> <li> 清洗缓冲区的准备 <ol> <li> 带来20x 和 #160; 洗涤缓冲器 (20x 和 #160;P bs 与 1% Tween-20) 提供的套件, 以室温和漩涡, 使所有的盐溶液. </li>
		<li> 稀释25毫升的20x 和 #160; 洗涤缓冲器与475毫升去离子水。将未使用的部分存储在2和 #176 之间; c 和8和 #176; c 最多为一个月. </li>
	</ol> </li> <li> 准备矩阵 B (仅用于血清和血浆样品) <ol> <li> 添加5.0 毫升的化验缓冲液 (PBS 与 1% BSA) 包括在装有冻干基质的瓶子中 b. 允许至少15分钟完成 reconstitution, 然后漩涡混合好。剩余的重组矩阵 B 可以存储在和 #8804;-70 和 #176; C 最多为一个月. </li>
	</ol> </li> </ol> 3。标准准备 注意: 此面板中的每个分析物的最高标准浓度为 1万 pg/毫升. 
<ol> <li> 添加250和 #181; 检测缓冲液以重建冻干小鼠 Th 细胞因子标准鸡尾酒。混合简单的涡流和允许小瓶坐在室温下10分钟. </li>
	<li> 将标准鸡尾酒转换为标签和 #34 的聚丙烯离心管; C7 和 #34。这将被用作最高标准. </li>
	<li> 将6聚丙烯离心管标记为 C6、C5、C4、C3、C2 和 C1. </li>
	<li> 添加75和 #181; 对每一个管子的化验缓冲器. </li>
	<li> 传输25和 #181; 顶部标准的 L C7 C6 管, 并涡流混合均匀。这将是 C6 标准. </li>
	<li> 继续执行串行1:4 稀释, 方法是使用新的吸管提示为每个管道添加25和 #181; 前一个标准到75和 #181; l 在下一最低标准管中检测缓冲器, 其次为涡流以获得标准 C5、C4、C3、C2 和 C1。使用化验缓冲器作为 0 pg/mL 标准 (C0). </li>
</ol> 4。示例稀释 注意: 使用此方法进行多稀释的初步试验可能需要确定特定生物样本集的最适当稀释因子。适当的稀释因子将产生浓度计算的样本, 在标准曲线的界限内。以下步意味作为指南并且可能必须根据样品类型经验主义地被确定. 
<ol> <li> 稀释血清或血浆样品2倍, 用化验缓冲液 ( e. g . 在聚丙烯离心管中, 稀释50和 #181; 50 和 #181;
	注: 如果需要进一步稀释样品, 稀释应用基质 B 代替化验缓冲器, 以确保精确测量。在不稀释的情况下加入血清或血浆样本会导致低的化验准确度, 并可能堵塞滤板。基质 B 是由聚集的小鼠血清内源性检测靶点的衰竭。它被用作血清或血浆样品稀释剂, 以避免基质效应, 这是已知的影响分析灵敏度的免疫. </li>
	<li> 测试细胞培养上清样品而不稀释. 
	注意: 分析物的水平可以有很大的差异, 从样品到样品。如有必要, 用新鲜的制备相应的细胞培养基或化验缓冲液稀释上清样品. </li>
</ol> 5。检测过程 注意: 该方法可以在聚丙烯滤板、微动管、或 V 底板中进行。该过滤板的检测程序是推荐的, 由于良好的样品样品的一致性, 化验的鲁棒性, 和易于处理。这一程序需要一个真空过滤单元洗涤 (由 end-user 提供). 
<ol> <li> 允许所有试剂在使用前对室温 (20-25 和 #176; C) 进行预热. </li>
	<li> 在检测设置和孵化步骤期间, 始终将过滤板设置在倒置的板盖上, 以便板的底部不会接触任何表面, 因为它可能会导致泄漏. </li>
	<li> 在整个检测过程中保持板直立, 包括清洗步骤, 以避免丢失珠子. </li>
	<li> 将盘子放在黑暗中或用铝箔包裹所有孵化步骤. </li>
	<li> 将所有标准和示例作为重复运行, 按顺序排列在盘上. </li>
	<li> 预湿过滤板, 加入100和 #181; L 1x 和 #160; 每个井清洗缓冲器, 并让它在室温下坐1分钟 (如果使用过滤器底部微). </li>
	<li> 使用真空歧管 (5-十年代) 删除缓冲卷。不超过 10 #34; 真空汞。涂抹过量洗涤从盘子底部的缓冲器上按下盘子上的一叠干净的纸巾。把盘子放在倒置的盘子盖上. 
	注: 如果使用 V 底微或微动管电泳仪进行检测, 则可省略步骤 5.1-5.2. </li>
	<li> 对细胞培养上清液样品, 加入25和 #181; 对所有井进行化验缓冲。在标准井中加入25和 #181;在样品井中添加25和 #181; L. </li>
	<li> 用于测量血清或血浆样品, 在标准井中加入25和 #181;在样品井中加入25和 #181;在标准井中加入25和 #181;在样品井中添加25和 #181; 每个稀释的血清或血浆样品的 L. </li>
	<li> 漩涡混合珠三十年代. 添加25和 #181; 每个井的混合珠, 摇动珠瓶间歇, 以避免珠沉淀。最后的音量应该是75和 #181; 我在每个井后添加珠子. </li>
	<li> 用印版封口封口。把整个盘子包起来, 包括倒置的板盖, 用铝箔。将板放在一个平板振动筛上, 将其固定, 并在室温下以大约 500 rpm 摇动2小时。为防止泄漏, 请勿向压板封口机施加正压. </li>
	<li> 不倒置, 将板放在真空流形上, 并像以前一样应用真空, 并添加200和 #181; 1x 和 #160 的 L; 每个井的洗涤缓冲器. </li>
	<li> 通过真空过滤去除检测板井的内容。用吸水垫或纸巾在盘子底部涂抹多余的洗涤缓冲器。重复此步骤一次. 
	注: 如果使用 V 底微或微动管电泳法进行检测, 则跳过步骤5.12 和5.13。相反, 在室温下用 1000 x g 离心板5分钟, 然后用多通道吸管除去上清液. </li>
	<li> 添加25和 #181; L 检测抗体 (请参阅材料表). </li>
	<li> 用保鲜板封口。把整个盘子包起来, 包括倒置的板盖, 用铝箔. </li>
	<li> 在室温下将板放在一个平板振动筛上, 在大约 500 rpm 处摇动1小时. </li>
	<li> 无吸尘, 添加25和 #181; L 的 SA PE 试剂直接对每一个井。不需要稀释试剂. </li>
	<li> 用保鲜板封口。把整个盘子包起来, 包括倒置的板盖, 用铝箔. </li>
	<li> 将板放在平板振动筛上, 在室温下以大约 500 rpm 摇动30分钟. </li>
	<li> 重复上面的步骤 5.13 </li>
	<li> 添加200和 #181; L 1x 和 #160; 每个井清洗缓冲器。重新悬挂在平板振动筛上的珠子1分钟 </li>
	<li> 使用多通道吸管, 将样品从过滤板转移到流式细胞仪上读取样品. 
	注: 样品量可从200和 #181 增加; l 到300和 #181; l 通过添加额外的100和 #181; l 1x 和 #160; 每管清洗缓冲器, 避免样品运行干燥。如有必要, 可将样品储存在4和 #176; C 保护不受光照, 并在第二天进行分析。但是, 长时间的采样存储会导致信号减少. </li>
</ol> 6。流式细胞仪设置 注意: 为了生成可靠的数据, 必须在数据采集前正确设置流式细胞仪。这一过程会因个别用户的不同而有所不同, 这取决于化验所执行的特定仪器的配置。下面讨论了一种能够测量 PE 和 APC 并配备488和 633 nm 激光器的细胞仪的广义设置过程. 
<ol> <li> 根据制造商和 #39 提供的指示, 启动流式细胞仪和采集软件. </li>
	<li> 在 Y 轴的 X 轴和 SSC (侧面散射) 上创建带有 FSC (正向散射) 的点图。将 FSC 和 SSC 设置为线性模式. </li>
	<li> 在 Y 轴的 X 轴和 APC 上创建一个带有 PE 的第二个点图。此绘图应设置为基于显示模式. </li>
	<li> 漩涡瓶的原始珠子包括在套件三十年代 re-suspend 的珠子。这些珠子含有一个内部 APC 染料, 由两个大小的人口组成. </li>
	<li> 将400和 #181 的原始珠子转到新的资产管制管. </li>
	<li> 将流式细胞仪流率设置为低. </li>
	<li> 运行未加工的珠子, 仔细调整 FSC 和 SSC 的增益和电压, 使这些珠子的大小群体都可以明显地分开, 容易被浇口. </li>
	<li> 调整 FSC 阈值以排除不需要的事件 ( 即 碎片或气泡). </li>
	<li> 在 FSC 与 SSC 的剧情中, 画一扇包括所有珠群的门. 
	注: 原珠有两个大小的珠子的数量, 小和 #34; 珠子和 #34; 大 #34; B 珠和 #34; 区域. </li>
	<li> 在 X 轴和 Y 轴上的 APC 上显示带有 PE 的第二个点图上的从 FSC 与 SSC 绘制的封闭珠群. </li>
	<li> 调整 apc 荧光通道的 PMT 电压, 以便所有珠群的 apc 信号都有一个介于 1 x 10 1 和 5 x 10 3 之间的中值荧光强度. </li>
	<li> 漩涡小瓶 PE 设置珠三十年代 re-suspend 珠. </li>
	<li> 将400和 #181 的 PE 珠转到新的资产管制管. </li>
	<li> 用 PE 珠管从流式细胞仪中更换原珠管. </li>
	<li> 调整 pe 荧光通道设置的光电倍增管 (PMT) 电压, 使 pe 微珠的多品种在 pe 小瓶上列出的具体范围之间. 
	注: PE 设置珠包含一个人口大小的珠子 (和 #34; 一个珠子和 #34; 只). </li>
</ol> 7。数据获取 注意: 与在给定仪器上获取数据相关的确切过程可能会有所不同, 并且依赖于细胞仪和 #39; s 配置规范和所使用的接口软件。因此, 下面的说明旨在强调在化验中所需采取的步骤, 而不考虑在化验中使用的细胞仪. 
<ol> <li> 验证细胞仪流率是否仍设置为低. </li>
	<li> 将要获取的珠子事件数设置为每个分析物的约300。对于一个13丛的面板, 这等同于获得3900事件结合从两个珠子大小的人口 (a + B 珠). </li>
	<li> 在分析之前将每个样本5秒涡旋. </li>
	<li> 读取示例。在读取样本时, 先将流式细胞仪设置为设置模式, 然后再切换到采集模式后再进行微珠种群的稳定. </li>
	<li> 对数据文件使用具有连续编号的简单名称以方便数据分析. </li>
	<li> 只导出封闭事件 (A + B 珠区域), 而不是总事件. </li>
	<li> 将所有 FCS 文件存储在每个检测的同一文件夹中。如果运行多个分析, 请为每个化验创建一个单独的文件夹. </li>
</ol> p 类= "jove_title" > 8。继续进行数据分析 注意: 在流式细胞仪上生成的 FCS 文件应该使用数据分析软件进行分析, 可以免费下载 14 。
<ol> <li> 在 PC 上安装数据分析软件. </li>
	<li> 将所有检测 FCS 文件传输到包含分析软件的计算机上. </li>
	<li> 将许可证密钥转换器 (包括在套件中) 插入计算机的 USB 端口. </li>
	<li> 启动数据分析软件. </li>
	<li> 单击蓝色和 #34; 添加文件和 #34; 按钮位于屏幕顶部. </li>
	<li> 在显示的弹出窗口中导航到包含 FCS 文件的文件夹. </li>
	<li> 单击并将所有检测 FCS 文件从弹出窗口拖到软件显示中。所有文件现在都应显示在列表中. </li>
	<li> 单击绿色和 #34; 下一 #34; 显示器右下角的按钮。左键单击并按住将小蓝标准曲线按钮 (C7 到 C0) 拖动到列表中相应的 FCS 文件中, 以定义标准曲线. </li>
	<li> 单击绿色和 #34; 下一 #34; 右下角的按钮, 打开弹出窗口. </li>
	<li> 在门控窗口的左侧输入 A 和 B 珠区域的名称检测分析物目标及其关联的磁珠 ID (在随化验提供的手册中找到) 按升序排列. </li>
	<li> 从弹出窗口的顶部选择 "浇口" 工具, 并使用它在 FSC 和 SSC 图中绘制2扇门, 一颗珠子绕一圈, 一秒钟围绕 B 珠. </li>
	<li> 查看显示在 FSC 与 SSC 图中的 APC 与 PE 散射图形。将有6分析物带在珠 (左图) 和7分析物带在 B 珠子 (正确的剧情). 
	注意: 通过选择顶部的橡皮擦工具并单击删除给定的门, 可以手动重绘门。然后, 可以选择浇注工具, 并用于手动将门应用于所需的带区. </li>
	<li> 单击 "绿色和 #34"; "确定" 和 "#34; 按钮关闭弹出窗口并返回到 FCS 文件的列表. </li>
	<li> 通过右键单击给定文件、选择和 #34;D ilution 折叠和 #34 来定义对生物样本进行的任何稀释; 从菜单中输入正确的值. </li>
	<li> 点击绿色和 #34; 运行和 #34; 在显示屏右下角的按钮, 以生成测试的标准曲线并计算未知生物样品的浓度. </li>
</ol>

<ol>
	<li>Stanley, A. C., Lacy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+for+cytokine+secretion.">Pathways for cytokine secretion.</a> Physiology (Bethesda). 25, 218-229 (2010).</li><li>Nicola, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+pleiotropy+and+redundancy:+a+view+from+the+receptor.">Cytokine pleiotropy and redundancy: a view from the receptor.</a> Stem Cells. 12, (1994).</li><li>Miyajima, A., Hara, T., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+subunits+of+cytokine+receptors+and+the+functional+redundancy+of+cytokines.">Common subunits of cytokine receptors and the functional redundancy of cytokines.</a> Trends Biochem Sci. 17, 378-382 (1992).</li><li>Costantini, S., Castello, G., Colonna, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Cytokinome:+a+new+challenge+for+systems+biology.">Human Cytokinome: a new challenge for systems biology.</a> Bioinformation. 5, 166-167 (2010).</li><li>Capone, F., 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=Serum+Cytokinome+Profile+Evaluation:+A+Tool+to+Define+New+Diagnostic+and+Prognostic+Markers+of+Cancer+Using+Multiplexed+Bead-Based+Immunoassays.">Serum Cytokinome Profile Evaluation: A Tool to Define New Diagnostic and Prognostic Markers of Cancer Using Multiplexed Bead-Based Immunoassays.</a> Mediators Inflamm. , 3064643 (2016).</li><li>Schett, G., Elewaut, D., McInnes, I. B., Dayer, J. M., Neurath, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+cytokine+networks+fuel+inflammation:+Toward+a+cytokine-based+disease+taxonomy.">How cytokine networks fuel inflammation: Toward a cytokine-based disease taxonomy.</a> Nat Med. 19, 822-824 (2013).</li><li>Tse, E., Kwong, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+lymphoma:+Microenvironment-related+biomarkers.">T-cell lymphoma: Microenvironment-related biomarkers.</a> Semin Cancer Biol. 34, 46-51 (2015).</li><li>Reale, M., Greig, N. H., Kamal, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+chemo-cytokine+profiles+in+Alzheimer's+and+Parkinson's+diseases.">Peripheral chemo-cytokine profiles in Alzheimer's and Parkinson's diseases.</a> Mini Rev Med Chem. 9, 1229-1241 (2009).</li><li>Vistnes, M., Christensen, G., Omland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+cytokine+biomarkers+in+heart+failure.">Multiple cytokine biomarkers in heart failure.</a> Expert Rev Mol Diagn. 10, 147-157 (2010).</li><li>Gobel, 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=Blood+coagulation+factor+XII+drives+adaptive+immunity+during+neuroinflammation+via+CD87-mediated+modulation+of+dendritic+cells.">Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells.</a> Nat Commun. 7, 11626 (2016).</li><li>Palm, A. K., Friedrich, H. C., Kleinau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+marginal+zone+B+cells+in+mice:+a+novel+subset+with+dormant+self-reactivity.">Nodal marginal zone B cells in mice: a novel subset with dormant self-reactivity.</a> Sci Rep. 6, 27687 (2016).</li><li>Yu, Y., 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=The+transcription+factor+Bcl11b+is+specifically+expressed+in+group+2+innate+lymphoid+cells+and+is+essential+for+their+development.">The transcription factor Bcl11b is specifically expressed in group 2 innate lymphoid cells and is essential for their development.</a> J Exp Med. 212, 865-874 (2015).</li><li>Pelly, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-4-producing+ILC2s+are+required+for+the+differentiation+of+TH2+cells+following+Heligmosomoides+polygyrus+infection.">IL-4-producing ILC2s are required for the differentiation of TH2 cells following Heligmosomoides polygyrus infection.</a> Mucosal Immunol. 9, 1407-1417 (2016).</li><li>. LEGENDplex Multiplex Assays Available from: <a target="_blank" href="https://www.biolegend.com/legendplex">https://www.biolegend.com/legendplex</a> (2017)</li><li>Autieri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-+and+anti-inflammatory+networks+in+atherosclerosis.">Pro- and anti-inflammatory networks in atherosclerosis.</a> ISRN Vascular Medicine. 2012, (2012).</li><li>West, N. 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=Emerging+cytokine+networks+in+colorectal+cancer.">Emerging cytokine networks in colorectal cancer.</a> Nat Rev Immunol. 15, 615-629 (2015).</li><li>Becher, B., Spath, S., Goverman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+networks+in+neuroinflammation.">Cytokine networks in neuroinflammation.</a> Nat Rev Immunol. 17, 49-59 (2017).</li></ol>

作者受雇于 BioLegend, 它制造了本手稿中所描述的试剂。Vigene 技术, Inc. 开发了 LEGENDplex 数据分析软件, 用于分析从本手稿中所描述的化验数据。

本协议所描述的检测格式可以提供生物样品中可溶性介质的定量, 只要实验者有良好的实验室技术, 并遵守推荐的协议。
为了保证可靠的结果, 必须遵循几个关键步骤。首先, 必须允许重组的标准, 在化验缓冲区的全部时间建议, 并在只稀释后, 聚丙烯管。聚苯乙烯管可以促进蛋白质粘附在管子的壁上, 在生成标准曲线时会导致低信号。第二, 在孵化过程中适当的晃动是至关重要的。没有适当的搅拌, 检测珠子的结合特性就会受到严重的阻碍。此外, 还需要在孵化步骤之间进行适当的冲洗。实验者必须确保在开始下一步的协议之前从油井中除去多余的试剂。仪器设置的流式细胞仪用于审问的化验珠子, 必须优化, 以及。特别是, PMT 设置必须调整, 以确保正确的珠分离和广泛的动态范围的标准曲线。最后, 在对细胞仪进行分析之前, 必须 vortexed 珠, 以确保适当的悬浮和避免形成的骨料, 可以扭曲的结果。
该协议可能会被修改, 以分析组织匀以及本手稿中讨论的样本类型, 只要研究员愿意优化他们的裂解协议之前, 执行大型, 全板的化验。实际的技术当然会因组织类型的不同而有所不同;然而, 一般来说, 该协议应使用一个中性的 pH 缓冲, 其中含有离子盐的生理浓度, 没有变性化学品, 最好没有洗涤剂。如果必须使用洗涤剂, 非离子洗涤剂浓度应保持在最低限度 (如不超过 1%)。该缓冲液还应含有足够的蛋白酶抑制剂, 以防止靶蛋白水解降解。无论使用何种裂解协议, 最后的准备工作都应离心, 以便在分析之前去除微粒。
关于化验的限制, 生物样品类型中分析物靶点的浓度可能有很大差异。为了正确地量化分析物的浓度, 它们必须属于标准曲线的顶部和底部值。对曲线值的推断不一定可靠, 也不推荐。因此, 研究者必须在进行 full-plate 试验之前, 在试验中优化稀释的样品。此外, 仔细准备要化验的生物样品对这种化验格式的成功至关重要。lipemic、溶血或含有蛋白质碎片的样品会影响抗原抗体相互作用, 从而确定免疫分析的核心原理, 并呈现出 uninterpretable 的结果。
对于现有的量化方法, 由于多种原因, 这种检测具有重要意义。当正常运行时, 检测格式可以定量13分析从一个样本使用远远小于需要分析样品使用传统的 ELISA 测定。这种化验格式也不需要专门的仪器, 以执行。这是一个优势相比, 其他商业可用的 bead-based 免疫, 因为化验可以运行使用任何数量的常用流 cytometers。
科学探究分泌因子和细胞因子网络在健康和疾病中所起的作用正在扩大。这篇手稿中描述的化验格式可以成为研究人员了解这些复杂现象的重要工具。积极的生物医学研究计划正在开始探索细胞因子网络的作用, 不仅在诸如广泛性炎症6, 而且在特定的疾病状态, 如动脉粥样硬化, 癌症和 neuroinflammation 的背景下,15,16,17。毫无疑问, 调查将继续在这些领域增长, 因为寻找治疗这些慢性病的新疗法扩大。对与这些疾病相关的可溶性介质的准确和可靠的定量化的需要仍然是一个重要的研究重点。

作为可溶性信号分子的作用, 细胞因子介导了控制宿主免疫应答的高度协调和多因素过程。它们是在炎症过程的所有阶段, 从起始到解决, 并调节一个复杂的相互作用网络, 包括他们自己的合成和他们的细胞受体1。这种通信网络的复杂性超出了单个组件之间可能存在的协同或对抗性关系。事实上, 许多细胞因子是已知的共享冗余或至少部分重叠的功能2,3。
综合系统生物学方法, 同时量化多个细胞因子分析目前正在提供一个越来越全面的理解独特的 cytokinomes, 协调免疫反应的基础上多疾病状态4,5。这些疾病状态范围从普遍的炎症到癌症, 神经退化的情况和心血管疾病6,7,8,9。
这种细胞因子网络可以使用 LEGENDplex bead-based 免疫进行有效的审讯。这些化验是基于与夹心酶联免疫吸附法 (ELISA) 相同的原理, 并使用荧光编码微球与捕获抗体共价键连接到他们的表面。这些抗体固定在比传统 ELISA 格式所要求的表面积小得多的表面上。这使得这样的化验能够以更少的样本量进行, 同时减少非特异性结合, 并提供多分析的多路复用分析。
本协议中描述的检测方法利用这一技术来同时定量多达13目标。从这个分析数据可以得到使用广泛的各种常见的流动 cytometers, 不像其他可用的化验不需要使用专门的化验专用仪器。随着经过验证的分析物面板的扩展目录, 这些化验已被用于几个正在进行的生物医学研究项目10,11,12,13。
为了证明化验格式的易用性和实用性, 在本实验中, 我们使用小鼠 T 帮助细胞因子定量13分离细胞因子的浓度从组织培养清得到的小鼠脾培养下多刺激条件。除了组织培养清, 这种检测也可以使用血清或血浆样品进行。

<table>
	<tbody>
		<tr>
			<td>Allegra 6R Centrifuge with MICROPLUS Carrier Adaptor</td>
			<td>Beckman Coulter</td>
			<td>366816</td>
			<td>For LEGENDplex assays is run in microtubes, V-or U-bottom 96-well plate (optional)</td>
		</tr>
		<tr>
			<td>LEGENDplex Mouse T helper Cytokine Panel</td>
			<td>BioLegend</td>
			<td>740005</td>
			<td>Multiplex Immunoassay (13-plex)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD3&#949; antibody</td>
			<td>BioLegend</td>
			<td>100314</td>
			<td>Clone 145-2C11, low endotoxin, azide free format</td>
		</tr>
		<tr>
			<td>Anti-mouse CD28 antibody</td>
			<td>BioLegend</td>
			<td>102112</td>
			<td>Clone 37.51, low endotoxin, azide free format</td>
		</tr>
		<tr>
			<td>Vacuum Pump</td>
			<td>EMD Millipore</td>
			<td>WP6111560</td>
			<td>For LEGENDplex assays using filter plates (recommended)</td>
		</tr>
		<tr>
			<td>Vacuum Manifold</td>
			<td>EMD Millipore</td>
			<td>MSVMHTS00</td>
			<td>For LEGENDplex assays using filter plates (recommended)</td>
		</tr>
		<tr>
			<td>Sterile Disposable Reagent Reservoirs</td>
			<td>Fisher Scientific</td>
			<td>07-200-130</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Polypropylene MicroFACS Tubes</td>
			<td>Fisher Scientific</td>
			<td>11-842-90</td>
			<td>For LEGENDplex assays is run in microtubes, V-or U-bottom 96-well plate (optional)</td>
		</tr>
		<tr>
			<td>Pipette Kit</td>
			<td>Fisher Scientific</td>
			<td>14-388-100</td>
			<td>Four pipette sizes (0.2-2&#181;L, 2-20&#181;L, 20-200&#181;L, 100-1000&#181;L)</td>
		</tr>
		<tr>
			<td>Multi-Channel Pipette</td>
			<td>Fisher Scientific</td>
			<td>FA10011G</td>
			<td>capable of dispensing 5 &#956;L to 200 &#956;L</td>
		</tr>
		<tr>
			<td>Microplate Shaker</td>
			<td>Fisher Scientific</td>
			<td>88880023</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td>75002410</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fisher Scientific</td>
			<td>NC9885747</td>
			<td>12 x 75 mm round bottom</td>
		</tr>
		<tr>
			<td>PMA (Phorbol 12-myristate 13-acetate)</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS)</td>
			<td>Sigma-Aldrich</td>
			<td>L-8274</td>
			<td>Escherichia coli O26:B6</td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson B200 Ultrasonic Cleaner</td>
			<td>Sigma-Aldrich</td>
			<td>Z305359</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>Z666505</td>
			<td>1.5 mL polypropylene tubes</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>Various</td>
			<td>Various</td>
			<td>Cytometer equipped with single laser (488nm blue) or two lasers (488nm blue or 532nm green + 633nm Red) capable of reading emission wavelengths at 575nm and 660nm</td>
		</tr>
		<tr>
			<td>96-Well Polypropylene Plate</td>
			<td>VWR</td>
			<td>82050-662</td>
			<td>For LEGENDplex assays is run in microtubes, V-or U-bottom 96-well plate (optional)</td>
		</tr>
	</tbody>
</table>

multiplex cytokine profiling of stimulated mouse splenocytes using a cytometric bead-based immunoassay platform

Bead-based 免疫采用与三明治免疫相同的基本原理。捕获珠, 可以区分大小和内部复方 (APC) 荧光强度, 是共轭的抗体特有的特定分析物。接下来, 一个被定义的捕获珠集的选择面板孵化与一个生物样品包含目标分析特异的捕获抗体。化检测抗体鸡尾酒, 这导致形成捕获珠分析物检测抗体三明治。
最后, 加入亲和-藻 (SA PE), 它结合到化检测抗体, 提供荧光信号强度的比例与绑定的分析物的数量。利用流式细胞仪对分析物特定珠区的 PE 荧光信号进行量化, 并利用数据解析软件和该检测中生成的标准曲线确定特定分析的浓度。
在本实验中, 我们使用小鼠 T 帮助细胞因子的小组, 同时量化的浓度13分离细胞因子目标的组织培养清收集的小鼠脾在不同的刺激条件下培养。

该协议演示了如何使用 bead-based 免疫分析平台可以同时定量细胞因子的轮廓从生物样品使用流式仪。在这个例子中使用的生物样品是细胞培养清从老鼠脾以前在各种各样的活化条件下被孵化了, 虽然化验格式也被证实了与血清或血浆样品一起使用。
该方法生成的数据用于构造多路复用面板中所有分析的标准曲线。数据分析软件根据独特的珠粒大小和 APC 强度对每种特定的析因物进行分类, 并使用 PE 报告器对其进行量化。当在图 2A中的日志日志刻度上绘制时, 将演示动态范围以及检测的灵敏度。该软件采用分析数据和5参数曲线拟合算法, 不仅准确地计算用户提供的生物样品中的分析浓度, 而且还对所有标准曲线的高低端进行检测的极限 (表1).该协议中描述的格式还提供了高度健壮的检测精度。在图 2B和2C数据中, 描述了每个分析物的 intra-assay 可变性。两个单独的标准蛋白质浓度 (高和低) 被分析在一个化验与16复制每个样本。图 2C和2D表示目标分析的中间可变性来自三独立的检测。随着 intra-assay 精度实验的进行, 在每个独立实验中, 分别对高、低标准浓度进行了3复制。目标蛋白的计算浓度的最小变异性明显可见。
为了证明 bead-based 细胞测定在询问细胞因子表达谱中的效用, 小鼠脾在各种活化条件下孵育。除了刺激的控制, 细胞还孵育的 LPS, anti-CD3/anti-CD28 抗体, 或佛波 12-酸 13-醋酸盐和 ionomycin。细胞培养清收集48小时后, 使用小鼠 T 帮助细胞因子的小组检测。该实验的量化结果在图 3中进行了说明, 并清楚地描述了刺激条件对细胞因子浓度的影响。
上述结果是典型的, 只要实验者有良好的实验室技术, 并遵循提供的化验方案。这种化验格式的错误来源可能产生于潜伏期的偏差, 试剂体积, 洗涤, 或不正确的 PMT 设置的流式细胞仪用于分析样品。在图 4A中, 一个成功的分析的散点图显示了2清楚定义的明显的珠子群。这可以与图 4B进行对比, 在这里, 较差的洗涤和协议粘附导致了模糊两个种群的珠子聚合体。此外, 从左下象限的碎片可见, 总的细胞仪事件被导出进行分析, 而不是只用于门控事件的首选格式。正确的细胞仪设置是至关重要的, 以产生可靠的数据, 在这个化验。在图 4C中, 适当的 PMT 设置允许在 APC 分类通道中的每个6分析的分辨率。不正确的 PMT 设置将导致与图 4D中所示的数据类似, 其中 APC 通道具有具有重叠的分类强度的珠群。这将使化验无效, 因为如果没有明确定义的测定珠的数量, 就不可能计算分析物的浓度。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56440/56440fig2.jpg"> 
图 2: 标准曲线范围和检测精度.(a)使用鼠标 T 帮助器细胞因子面板生成的典型标准曲线。每种化验都必须运行标准曲线。(B-c)内部检测精度。分析了两种不同浓度靶蛋白 (高、低) 样品, 并对每个样本进行16次复制。数据表示为平均值 + 标准偏差。(D-E)间测定精度。两个样本的目标蛋白不同浓度 (高和低) 分析了三独立的化验和3复制每个样本。数据表示为平均值 + 标准偏差。<a href="//ecsource.jove.com/files/ftp_upload/56440/56440fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56440/56440fig3.jpg"> 
图 3: 代表性结果的量化.在各种情况下, 鼠标脾 (1 x 106单元格) 在37° c + 5% CO2中被培养: 刺激, LPS (100 ng/毫升), αCD3 (1 µg/毫升板涂层) + αCD28 (1 µg/毫升可溶性), PMA (20 ng/毫升) + Ionomycin (500 ng/毫升)。文化清收集后48小时, 然后量化使用鼠标 T 帮助细胞因子面板。不同的细胞因子表达谱对刺激条件的反应是显而易见的。<a href="//ecsource.jove.com/files/ftp_upload/56440/56440fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56440/56440fig4.jpg"> 
图 4: 成功与不成功的化验结果.(A)成功的化验将有不同的珠子群体的微球群体。(B)不成功的检测将会有较差的人口分离、珠子聚合和过多的收集事件。(C)成功的检测在分类荧光通道中有明确定义的强度, 易于门和量化。(D)不成功的检测将会有很差的分离和分类强度, 从而使多珠种群重叠, 使得量化困难。这些错误中的许多可以通过仔细地遵守化验协议来避免。<a href="//ecsource.jove.com/files/ftp_upload/56440/56440fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">标准曲线</td>
			<td colspan="2">灵敏度 (pg/毫升)</td>
		</tr>
		<tr>
			<td>分析</td>
			<td>拟合公式</td>
			<td>简历</td>
			<td>R2</td>
			<td>分钟.</td>
			<td>最大.</td>
		</tr>
		<tr>
			<td>干扰素-γ</td>
			<td>5-P. 日志 (4.43、14.40、0.54、9.11、0.01)</td>
			<td>1.77%</td>
			<td>0.99</td>
			<td>2。1</td>
			<td>18105。0</td>
		</tr>
		<tr>
			<td>IL-5</td>
			<td>5-P. 日志 (4.36、12.68、0.64、5.73、0.36)</td>
			<td>1.32%</td>
			<td>1</td>
			<td>1。9</td>
			<td>14514。0</td>
		</tr>
		<tr>
			<td>TNF-α</td>
			<td>5-P. 日志 (4.36、11.63、0.79、5.45、0.37)</td>
			<td>1.48%</td>
			<td>0.99</td>
			<td>1。9</td>
			<td>20109。0</td>
		</tr>
		<tr>
			<td>IL-2</td>
			<td>5-P. 日志 (4.46、12.62、0.78、5.07、0.34)</td>
			<td>1.34%</td>
			<td>1</td>
			<td>1。9</td>
			<td>25109。0</td>
		</tr>
		<tr>
			<td>IL-6</td>
			<td>5-P. 日志 (4.58、11.66、0.64、5.77、0.37)</td>
			</tr></tbody></table>td > 1.70%
			<td>0.99</td>
			<td>2。0</td>
			<td>7022。0</td>
		
		<tr>
			<td>IL-4</td>
			<td>5-P. 日志 (4.27、12.23、0.62、5.72、0.36)</td>
			<td>1.36%</td>
			<td>1</td>
			<td>2。0</td>
			<td>13273。0</td>
		</tr>
		<tr>
			<td>IL-10</td>
			<td>5-P. 日志 (4.67、11.65、1.20、6.28、0.61)</td>
			<td>1.81%</td>
			<td>0.99</td>
			<td>2。2</td>
			<td>5190。0</td>
		</tr>
		<tr>
			<td>IL-9</td>
			<td>5-P. 日志 (4.32、13.86、0.86、5.90、0.45)</td>
			<td>1.82%</td>
			<td>1</td>
			<td>1。9</td>
			<td>12602。0</td>
		</tr>
		<tr>
			<td>IL-17A</td>
			<td>5-P. 日志 (4.06、14.03、0.47、6.52、0.32)</td>
			<td>0.99%</td>
			<td>1</td>
			<td>2。0</td>
			<td>12285。0</td>
		</tr>
		<tr>
			<td>IL-17F</td>
			<td>5-P. 日志 (4.52、12.15、0.61、5.52、0.34)</td>
			<td>0.92%</td>
			<td>1</td>
			<td>2。0</td>
			<td>14841。0</td>
		</tr>
		<tr>
			<td>IL-21</td>
			<td>5-P. 日志 (4.90、8.03、1.77、4.74、1.11)</td>
			<td>0.79%</td>
			<td>1</td>
			<td>9。0</td>
			<td>12943。0</td>
		</tr>
		<tr>
			<td>IL-22</td>
			<td>5-P. 日志 (4.57、12.56、0.63、6.06、0.39)</td>
			<td>1.16%</td>
			<td>1</td>
			<td>2。0</td>
			<td>6408。0</td>
		</tr>
		<tr>
			<td>IL-13</td>
			<td>5-P. 日志 (4.40、11.44、0.22、3.19、1.45)</td>
			<td>1.11%</td>
			<td>1</td>
			<td>2。1</td>
			<td>16728。0</td>
		</tr>
	

表 1: 标准曲线拟合和检测灵敏度.数据分析软件被用来生成标准曲线的所有分析包含在多重检测。这一全自动分析应用了一个稳健的5参数曲线拟合算法的稀释系列标准从该试验, 也被用来定义的检测灵敏度使用的理论界限的探测。

Watch this Scientific Journal Video about Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform at JoVE.com

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform

该协议描述了多细胞因子靶点的定量, 同时在组织培养清收集的刺激小鼠脾使用多路微珠基免疫分析平台和流式仪。

细胞 Bead-based 免疫分析平台对受激小鼠脾多重细胞因子的研究

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal ...

multiplex-cytokine-profiling-stimulated-mouse-splenocytes-using

Research

JoVE Journal

Immunology and Infection

44.7K 次观看.  BioLegend. 该协议描述了多细胞因子靶点的定量, 同时在组织培养清收集的刺激小鼠脾使用多路微珠基免疫分析平台和流式仪。

视频: Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

科研

免疫与感染

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

侵染微生物利用数字条码平台的基因表达谱

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and protection to the underlying cells. Analysis of tears is an emerging area for the identification of biomarkers for the prediction, diagnosis, and prognosis of various ocular diseases. Tears are easily accessible and their collection is non-invasive. Therefore, advancing technologies are gaining prominence for identification of multiple analytes in tears to study changes in protein or metabolite composition and its association with pathological conditions. Tear cytokines are ideal biomarkers for studying the health of the ocular surface and also help in understanding the mechanisms of different ocular surface disorders like dry eye disease and vernal conjunctivitis. Bead based multiplex assays have the capability of detecting multiple analytes in a small amount of sample with a higher sensitivity. Here we describe a standardized protocol of tear sample collection, extraction and analysis of cytokine profiling using a bead based multiplex assay.

Analysis of tear film cytokines helps in studying various ocular diseases. Bead based multiplex assays are simple and sensitive and enable the testing of multiple targets in samples with small volumes. Here we describe a protocol for tear film cytokine profiling a using bead based multiplex assay.

基于磁珠复合法的撕裂细胞因子分析

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

利用广域跟踪平台对大型线虫种群进行自动行为分析

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Antibiotic Dereplication Using the Antibiotic Resistance Platform

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this challenge, dereplication, which is the process of identifying known compounds, is performed on samples of interest. Methods for dereplication such as analytical separation followed by mass spectrometry are time-consuming and resource-intensive. To improve the dereplication process, we have developed the antibiotic resistance platform (ARP). The ARP is a library of approximately 100 antibiotic resistance genes that have been individually cloned into Escherichia coli. This strain collection has many applications, including a cost-effective and facile method for antibiotic dereplication. The process involves the fermentation of antibiotic-producing microbes on the surface of rectangular Petri dishes containing solid medium, thereby allowing for the secretion and diffusion of secondary metabolites through the medium. After a 6 day fermentation period, the microbial biomass is removed, and a thin agar-overlay is added to the Petri dish to create a smooth surface and enable the growth of the E. coli indicator strains. Our collection of ARP strains is then pinned onto the surface of the antibiotic-containing Petri dish. The plate is next incubated overnight to allow for E. coli growth on the surface of the overlay. Only strains containing resistance to a specific antibiotic (or class) grow on this surface enabling rapid identification of the produced compound. This method has been successfully used for the identification of producers of known antibiotics and as a means to identify those producing novel compounds.

We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient.

使用抗生素耐药性平台进行抗生素脱除

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this ...

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

全鼠眼的消化，用于单核噬细胞的多参数流细胞学分析

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...