The authors thank Dr. M. Matrosovich for providing the parent MDCK and MDCK-SIAT1 cell lines and Roche Pharmaceuticals for supplying the oseltamivir carboxylate.We also appreciate Dr. Anne Weston for valuable suggestions on the manuscript. This study was dependent upon the valued collaboration of the WHO National Influenza Centres and the WHO CCs within the WHO GISRS, who provided the influenza viruses used. This work was funded by the Medical Research Council through Programme U117512723.

注:以下协议的生物安全水平（BSL）是季节性流感病毒和BSL 3+为潜在的流感大流行的病毒BSL 2。病毒滴定法需要事先中和实验来决定病毒控制（VC）的病毒浓度。 1.病毒滴度注意:根据病毒的数量和重复的次数，一孔板可以设置具有以下灵活性:（1）病毒稀释可以沿任一的行或列（ 图1）被布置。每个病毒应该占据一个独立的行/列。 （2）病毒稀释增量是柔性的，但它应与在左上角的最高病毒浓度开始。 （3）上有重复的数量没有限制。 注:的Madin Darby狗肾细胞（MDCK）和MDCK-SIAT1（SIAT）细胞好心由M. Matrosovich博士，马尔堡，提供芨芨草许多10。盟细胞是与人的CMP-N-乙酰神经氨酸稳定转染的MDCK细胞:唾液酸（SA）的增强表达β半乳糖苷α-2,6-唾液酸转移酶基因α2-6Gal终止寡糖。 <ol><li>等分部分足以MDCK细胞或MDCK-盟细胞8到96孔板（200μl/孔）。孵育细胞在37℃，5％CO 2的2或3天以达到融合。传播补充有10％热灭活胎牛血清（FCS）和抗生素（100U / ml青霉素和100μg/ ml链霉素）的DMEM细胞。孵育盟细胞在37℃，5％的CO 2和1mg / ml G418的硫酸盐。 注:稀释倍数取决于经验和所使用的细胞系。细胞单层必须汇合（即没有细胞壁或MDCK细胞和MDCK-SIAT1细胞紧密堆积布局可见轮廓）在病毒接种时。稀释液的例子基于MDCK或MDCK-SIAT1细胞汇合烧瓶的传代培养于表1中给出。 </li><li>清洗细胞3次，用200微升每孔病毒生长培养基（VGM）的。消毒用70％乙醇的多层洗板30分钟，然后冲洗在洗涤前的无菌磷酸盐缓冲盐水（PBS）或VGM。 </li><li>吸出VGM，立即加入50微升VGM的每个孔中。 </li><li>添加900微升VGM到每个6无菌试管中（或更少，这取决于测试的病毒和重复数）。吸取100微升病毒进入第一管，拌匀，并连续稀释，改变每个稀释度之间的提示。重复每个病毒滴定。 注意:这将给予10 -1病毒稀释至10-6。 </li><li>添加每种病毒稀释液50微升，起始于最高稀释度（1×10 -5 图1），在9重复的孔中（列9和10中的图1）6孔板中。 </li><li>放置在37℃的板2-3小时以使病毒感染的细胞。 注:A 2至3小时培养是必要的，以确保在接种病毒有足够的时间来达到单层。 </li><li>准备覆盖（10毫升/板） 注意:覆盖由5毫升的2×DMEM，胰蛋白酶（2微克/毫升的最终浓度），20微升的1毫克/毫升的库存，和5ml纤维素棉短绒（见材料清单）的。 </li><li>取出接种。 </li><li>加覆盖（200微升至每孔）。在37℃孵育过夜的，不受干扰。 注:病毒出芽发生之后大约4小时，因此重要的是，该板未在此时间后的干扰。 </li><li>吸从井中覆盖。 </li><li>加入冰冷的4％多聚甲醛的PBS（200微升/孔）。将它们放置在4℃下30分钟，或在室温下搅拌20分钟。 注:PBS A是自然pH值的磷酸盐缓冲盐水，10去˚F氯化钠0.25g的氯化钾，1.437克的 Na 2 HPO 4的0.25g的KH 2 PO 4，1升蒸馏水（见材料清单）。 注意:吸入和吞食可能造成灼伤时，多聚甲醛是有害的。还有具有致癌作用的证据有限，和皮肤接触后可能会引起过敏。 </li><li>吸出多聚甲醛，并用PBS洗涤板两次（200μl/孔）。 </li><li>存储在PBS A中的板在4℃下以供将来使用，或与下列步骤进行。 </li><li>添加透化缓冲液（100μl/孔）。离开它在室温下30分钟。 </li><li>用PBS洗两次板（200微升/孔）。 </li><li>加入50微升一抗，抗流感A型鼠标单克隆抗体（1:1,000 ELISA缓冲液），每口井。孵育它在室温下1小时（或4℃过夜），在摇动。 </li><li>在100μl洗涤缓冲液（0.05％吐温80在PBS中A，V / V）的p洗3次呃好。孵育它在室温下洗涤之间5分钟。另外，洗3次，而无需使用每孔300微升孵化。 </li><li>加入50μl的第二抗体的山羊抗小鼠IgG（H + L）HRP缀合物（1:1000在ELISA缓冲液），每孔。孵育它在室温下搅拌1小时，用摇动。 </li><li>如在步骤1.17中所述洗3次。 </li><li>添加底物（50μl/孔）。孵育，在室温下30分钟或直至蓝色的发展是清晰可见。 </li><li>停止反应，用200μl每孔蒸馏水洗涤平板两次，在室温下温育的洗涤之间2 -3分钟。 </li><li>空气干燥板，并将其存储在一个黑暗的地方（ 例如，包装在铝箔）。 </li></ol> 2.定量滴定<ol><li>放置在平板扫描仪的扫描区一个孔板， 如图2a所示。使用L形的位置限制确保最佳的和可重复的成像位置。 注意:可以将图像两个著名板在一次扫描。 </li><li>扫描图像。 注:设置示于表2中 。 </li><li>运行"多孔板阅读器"的软件来计算所需的病毒浓度（ 图3）。 <ol><li>点击"加载图像"按钮，加载图像。滑入"全球阈值"选项卡以调整采样阈值直方图的红条。点击"更新"按钮来检查图像上的效果。 </li><li>勾选"计算中和/滴定"框。点击"采集"按钮以量化的ICP。点击"保存"按钮，在出现提示时保存采样结果。 注:在采样过程后，如果"计算中和/滴定"框中打勾自动出现一个名为"中和滴定与计算"新窗口。 </li><li>负载或输入井板定义地图。表明在"滴定:最优病毒种群（％）"的阈值（ 例如，30％）。选择"滴定进程"选项卡来计算滴定结果。检查滴定结果。点击"保存并关闭"按钮，保存滴定结果。 注:请参阅补充S1有关软件的详细说明。可根据要求定制的软件的副本。 注:通常，最好的病毒稀释度为50％左右（20-85％）细胞总数的ICP斑块还原法产量每孔11之内。 </li></ol></li></ol> 3.病毒中和注:计算的中和测定所需板的数量。每个板可容纳一个病毒和一些抗血清。 注意:根据抗血清的数量和重复的次数，一孔板可以设置与第Ë以下灵活性:（1）血清稀释度可以沿任一行或一列（ 图4）被布置。每个血清应该占据一个独立的行或列。 （2）血清稀释度的增量是柔性的，但它应与在板的左上角的最高血清浓度开始。 （3）重复的数量平衡的抗血清的数量。更多的重复可以帮助理顺实验的变化。 （4）在VC的数量和单元控制（CC）的重复是柔性的，但它们也应该是在单独的行或列的数目。据预计，VC副本的数目比抗血清的显著更大。 <ol><li>制备细胞单层，如在步骤1.1-1.3，2或3天提前。 </li><li>加入50μlVGM的至板的各孔中。 </li><li>分别用11和12列的VC和CC。添加1:20受体破坏酶（RDE）处理血清的50微升等分试样到科拉姆的第一行（A）的NS 1-10。 注:对于每种病毒和血清结合，该试验一式两份进行，从而一个板可，例如，包含一种病毒对五种抗血清进行测试。 </li><li>执行从A行从行H.转移50微升到行H（列1-10 图4），并丢弃50微升2倍系列稀释</li><li>加入50μl稀释液向CC柱（ 图4中第12列）的各孔中。 </li><li>加入50μl病毒至板的各孔中，除了用于CC柱（列1-11，行AH中图4）。 </li><li>孵育它在37℃下2-3小时。取出接种。加覆盖（200微升）到每个孔中。过夜孵育它在37℃下，原状。修复和染色板作为病毒滴度（步骤1.11-1.22）。 </li></ol> 4.中和定量<ol><li>放置一个孔板在平板扫描仪的扫描区，如图Figu重新2A。使用L形位置的限制，以保证最佳的和可重复的成像位置。 注意:可以将图像两个著名板在一次扫描。 </li><li>扫描板，如在步骤2.2中描述。 </li><li>运行"多孔板阅读器"的软件来计算所需的病毒滴度（ 图3，步骤2.3）。 <ol><li>点击"加载图像"按钮，加载图像。滑动"全球阈值"的红色条及时调整采样阈值。点击"更新"按钮来检查的效果。 </li><li>勾选"计算中和/滴定"框。点击"采集"按钮以量化的ICP。点击"保存"按钮，在出现提示时保存采样结果。 注:在采样过程后，如果"计算中和/滴定"框中打勾自动出现一个名为"中和滴定与计算"新窗口。 </li><li>负载或输入井-P晚地图。指示阈值（ 例如，50％）"中和:感染扣除（％）"。 </li><li>选择"中和过程"来计算的滴度。检查滴度，并点击"保存并关闭"按钮，保存中和结果。 注:中和被报告为与预定的ICP还原（如50％或80％）对在VC（11栏的图4中的平均值）的血清的最高稀释度的倒数。 50％的减少ICP是在代表性的成果使用。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这项研究中，我们所描述的基于成像的MN测定以量化病毒之间的真实抗原关系。与其它噬斑减少测定法相比，该方法强调测量整个井的ICP，确保感染人口的完全覆盖。其斑块形成的独立性也加宽了测定，以能够形成主要隐形小斑块或只能管理单细胞感染病毒的应用。因此，该测定能够检查更多的病毒和更宽范围的抗体比HI的的影响，并且它有助于更全面地反映抗原相似或病毒12之间的差异。例如，当包括奥塞米韦羧酸盐中，MN测定较少受NA依赖性影响结合，反映抗原的差异更准确。在测定法的发展，大作出了努力以提高实验一致性，速度和检测敏感性，包括通过定量滴定，成像高通量与平板扫描仪输入控制病毒，并实现用户友好的数据处理平台。结果一般都符合并证实了这些HI的。值得注意的是，调查结果还显示所造成的文化选择的或偶发性的变化抗原性改变近期A和B型疫苗病毒抗原漂移变体之间抗原的差异，以及，如在某些A（H1N1）pdm09病毒HA1的G155E替换12。 该协议与相匹配的，给了最强的感染，这大大提高了成像信号的细胞系的选择病毒类型或亚型。实验变异用与测试抗血清的数量平衡重复的设计平滑。的不确定性也可以来自图像质量，这主要是由成像设备的影响。一个体面的彩色平板扫描仪可以显ficantly提高图像的对比度和降低噪音。输入病毒的中和量必须提前定量测定从相应的滴定而病毒仍然保持相似的状态。期间中和，抗血清反应的感染是针对基准病毒（VC）和背景水平（CC）之间的差归一化。 VC和CC的精确测量是中和实验必不可少的。建议更多的重复（一式八份分别在代表性的成果使用）。最后，了解软件是一个实验的成功至关重要。该软件已经过测试，在世卫组织流感中心，英国常规病毒特征进行了两年多。用于处理各种情况的详细说明在补充S1中提供的。 该MN测定法是适合于将样品覆盖一个电话分机的应用remely大视场，但只需要适度的成像分辨率。低成本和简单的设置，使系统随时提供给最病毒学实验室。在相同样品的测量结果的变化小于3％，这大致定义了扫描11中引入的不确定性。这种重复性是在许多病毒学研究的足够，并且可以通过平均从多个扫描图像来进一步降低。 总之，这项研究表明，在抗原性研究中经常使用，并支持目前流通的流感病毒，特别是对病毒列入人类流感疫苗的一年两次的选择详细的分析数据HI强大MN检测。

微中和（MN）测定法在病毒学用于中和抗体的定量和的抗病毒活性。作为血凝抑制的替代（HI）测定，MN测定法可以克服由受体结合于流感病毒的亲和力的改变，其可以变得复杂的HI的解释结果1,2,3-影响无抗原性的影响。直到最近，大多数MN测定法基于细胞病变效应（CPE）或酶联免疫吸附测定（ELISA）4,5。根据焦点和斑块减少MN试验于1990年6,7,8开发的。噬斑减少测定依赖于计数可见斑量化感染。然而，视觉计数仅覆盖大斑是由人眼可分辨的，尽管大多数斑很小，对于许多当前流行的病毒不可见的。这种不完整的覆盖面可能会导致考官之间和实验之间显著的变化，导致我ncomparable结果。另外，也不能使用该方法时，一些病毒表明单个细胞或非常小的斑块流产感染。 计数分辨率差可以通过引入基于成像的协议来改进。随着技术，光学显微镜和高通量的进步孔板读者可以提供准确的方法来计算感染的细胞9。下一个反式照明显微镜，具有标记某些标记感染的细胞可以通过在亚细胞分辨率的吸收或荧光对比度可视化。样品然后可以在电脑屏幕上进行分析。 不幸的是，由于在视场的限制，超过一百平铺图像，需要以覆盖单个孔。判断的板96孔将需要一万左右的图像的摄像处理。这种费力的过程是耗时和昂贵的，并且所获得的分辨率是在属升不必要对病毒感染的常规鉴定。预算有限的实验室可能会发现周围的平板扫描仪内置的方法提供了一种高性价比，高通量的选择。 在本文中，我们描述一种改进的空斑减少测定法MN是适合于大量的病毒的抗原特征和用于定量测定抗病毒活性和中和抗体。该试验具有几个优点:第一，它是一个基于成像的检测，能够测量在细胞水平的病毒感染，无论斑块大小的。内一个井计数总感染的细胞群（ICP）大大增加了检测灵敏度，使得有可能以低感染性表征病毒。其次，更准确地定量滴定之前中和引入到确定输入病毒的量。量化输入病毒显著降低杂物不同的实验之间化，使结果实验室之间更具可比性。第三，中和滴度可以直接通过分析图像，使得定量快速和方便用户的确定。最后，该协议提供了一种具有成本效益和高通量的替代与所需的分辨率和精度。定量是基于一个平板扫描仪和免费数据处理软件。整个安装占地面积小，是大多数实验室部署。 本文提出的协议包括四个主要步骤，包括病毒滴定法，滴定定量，病毒中和，中和定量。病毒滴定是制备实验确定在中和中使用的输入病毒的量。期间滴定，一些病毒浓度在一个96孔板施加到细胞单层。被感染的细胞，然后在第2，病毒定量DILU化，产生20％-85％ICP被依次施加作为输入病毒到相应的中和 第3节中和协议的效价是使用第4节实验证明遵循上述协议是在代表结果提出量化。该法已在过去两年，大多数目前流行的流感病毒，如A（H1N1）pdm09，A（H3N2）和乙型流感病毒彻底的测试。流感病毒鉴定结果已列入给了流感疫苗南半球在2016年和北半球2016至2017年使用建议世界卫生组织协商会议的报告。

<table border="0" cellpadding="0" cellspacing="0" width="1115">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:177px;">VGM</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:383px;">D6429, P0781</td>
			<td style="width:383px;">500 ml DMEM+5 ml Pen/Strepb</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:177px;">PBS A</td>
			<td style="width:172px;"></td>
			<td style="width:383px;"></td>
			<td style="width:383px;">Nature pH Phosphate-buffered saline: NaCl -10 gm, KCl - 0.25 gm, Na2HPO4 - 1.437 gm, KH2PO4 &#8211; 0.25 .gm, and Dist. Water &#8211; 1 L.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:177px;">Avicell</td>
			<td style="width:172px;">FMC</td>
			<td style="width:383px;">RC-581F</td>
			<td style="width:383px;">2.4g in 100ml distilled water dissolved by agitation on a magnetic stirrer for 1 hr. Sterilize by autoclaving.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:177px;">2XDMEM</td>
			<td style="width:172px;">Gibco&#160;</td>
			<td style="width:383px;">21935-028</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:177px;">Trypsin</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:383px;">T1426</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:177px;">Overlay (10ml/plate):</td>
			<td style="width:172px;"></td>
			<td style="width:383px;"></td>
			<td style="width:383px;">5ml 2X DMEM, Trypsin 2&#956;g/ml final conccentration,Avicell&#160; - 5ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:177px;">Triton X- 100e</td>
			<td style="width:172px;">Sigma&#160;</td>
			<td style="width:383px;">T8787</td>
			<td style="width:383px;">Permeabilisation buffer: 0.2% in PBS A (v/v)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:177px;">Tween 80</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:383px;">P5188</td>
			<td style="width:383px;">&#160;Wash Buffer: 0.05% Tween &#8211; 80 in PBS A (v/v)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:177px;">Horse serum</td>
			<td style="width:172px;">PAA Labs Ltd</td>
			<td style="width:383px;">B15-021</td>
			<td style="width:383px;">ELISA Buffer: 10%&#160; in PBS A (v/v) + 0.1% Tween 80</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:177px;">Mouse MAb against influenza type A</td>
			<td style="width:172px;">Biorad</td>
			<td style="width:383px;">MCA 400</td>
			<td style="width:383px;">1st Antibody: 1:1000 in ELISA Buffer</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:177px;">Goat anti-mouse IgG (H+L) HRP conjugate</td>
			<td style="width:172px;">Biorad</td>
			<td style="width:383px;">172-1011</td>
			<td style="width:383px;">2nd Antibody: 1:1000 in ELISA Buffer</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:177px;">True Blu peroxidase substrate</td>
			<td style="width:172px;">KPL&#160;</td>
			<td style="width:383px;">50-78-02</td>
			<td style="width:383px;">Substrate:&#160; True blue +0.03% H2O2g (1:1000 of 30% solution)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:177px;">96-well flat-bottom microtitre plates</td>
			<td style="width:172px;">Costar&#160;</td>
			<td style="width:383px;">3596</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:177px;">8 channel multiwall-plate washer and manifold</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:383px;">M2656</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:177px;">Perfection Plate Scanner</td>
			<td style="width:172px;">Epson</td>
			<td style="width:383px;">V750 Pro</td>
			<td style="width:383px;"><a href="http://www.epson.com/cgi-bin/Store/support/supDetail.jsp?oid=66134&amp;infoType=Downloads.">The imaging software can be downloaded freely from http://www.epson.com/cgi-bin/Store/support/supDetail.jsp?oid=66134&#38;infoType=Downloads.</a></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:177px;">Software operational environment: LabVIEW</td>
			<td style="width:172px;">National Instruments Corporation</td>
			<td style="width:383px;">Window based with NI Vision builder</td>
			<td style="width:383px;">Version: LabVIEW2012 or above</td>
		</tr>
	</tbody>
</table>

optimization of a quantitative micro-neutralization assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

经上述程序，我们提出最近的抗原特性的实验的一些结果。滴定设置旨在探讨八个输入病毒，与每个稀释度双重复。病毒稀释液进行试验1.0和1.0之间×10 -1×1.0 -6覆盖病毒之间的变化。测试病毒是由H3N2亚型，包括A /喀麦隆/ 15V-二千〇一十五分之三千五百三十八，A /符拉迪沃斯托克/ 36/2015年，A /莫斯科/ 103/2015年，A /托木斯克/ 5/2015 /，A /莫斯科/ 101 / 2015，A /莫斯科/ 100/2015，A /莫斯科/ 133/2015和A /布拉迪斯拉发/ 437/2015。滴定结果示于图5中示出。 图5d说明了的ICP的病毒稀释度的增加而减少。曲线进行归针对的一个井12内产生的所有小区感染同一病毒的ICP（定义为ICP饱和度）。如果ICP并没有随着喜达到饱和ghest病毒浓度，从相应的重复平均ICP代替（A /莫斯科/ 103/2015 图5d）。所产生ICP饱和的30％的病毒稀释液被选择作为输入的病毒稀释度中和（ 表3）。 中和的目的是有一个输入病毒对五种抗血清，对每个板双重复。所示的基准病毒是H3N2 A /斯德哥尔摩/ 63/2015五个抗血清A / HK14分之4801蛋F12 / 15，A / HK十四分之七千二百九十五MDCK F02 / 15，A /南非R2665 / 15 SIAT F50 / 15，A /瑞士十三分之九百七十一万五千九百二十三SIAT NIBF F18 / 15，和A /荷兰十四分之五百二十五SIAT F23 / 15。中和的结果在图6中示出。 图6d示出了具有血清稀释度的增加的感染的进展。在垂直轴上的归一化阳性群体代表的ICP的从对平均ICP相应的抗血清反应的比率的基准病毒12。未感染细胞对照背景ICP正常化过程中扣除。中和滴度被确定为对应于50％的ICP降低（ 表4）的抗血清稀释液的倒数。线性内插来估计滴度两个相邻的血清稀释度之间落下。 <img alt="图1" src="/files/ftp_upload/54897/54897fig1.jpg" /> 图1: 在病毒滴定实验一孔板设置举例。测试病毒被分配到不同的行（A至H）。列是针对不同的病毒稀释液（1到12）。两列被用作复制每个病毒稀释。比例尺= 10毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/54897fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2: 样品扫描系统的原理图。 （a）在平板扫描仪的成像位置A 96孔板（从再版 附图11用从爱思唯尔BV许可），和（b）中所示的L形位置极限尺寸（一）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/54897fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54897/54897fig3.jpg" /> 图3:"多孔板阅读器"定量软件的台式图。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/54897fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p&gt; <img alt="图4" src="/files/ftp_upload/54897/54897fig4.jpg" /> 图4: 在中和实验中孔板设置举例。各抗血清被分配给两列重复（1〜10）。行针对不同的血清稀释（A至H）。该病毒控制需要11列，有八个重复（A11至H11）。小区控制是在第12栏，有八个重复（A12至H12）。比例尺= 10毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/54897fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54897/54897fig5.jpg" /> 图5:滴定实验的结果。 （ 一 ）该井板设置，（ 二 ）扫描孔板图像，（C）说明的颜色编码的，定量，病毒感染的细胞群，和针对病毒稀释液（D）的归一化的病毒感染的细胞群。 30％的ICP用作阈值。 （d）中的误差棒是从样品重复（±1 SD）标准差（SD）。的（b）和（c）比例尺= 10毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/54897fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54897/54897fig6.jpg" /> 图6: 中和试验的结果。 （ 一 ）该井板设置，（ 二 ）扫描孔板图像，（c）该定量，病毒感染的细胞群的画像，和对血清稀释度（D）的归一化的病毒感染的细胞群。输入VIRUS（VC）是A /斯德哥尔摩/ 63/2015 50％的ICP来计算滴度。 （d）中的误差棒是从样品重复（±1 SD）标准差（SD）。的（b）和（c）比例尺= 10毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/54897fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 补充S1: <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/Supplementary_S1.pdf" target="_blank">请点击这里下载该文件。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="3"> 典型的稀释 </td></tr><tr><td></td><td> MDCK细胞 </td><td> MDCK-SIAT1细胞 </td></tr><tr><td> 2天 </td><td> 1:5（1 + 4） </td><td> 1:10（1 + 9） </td></tr><tr><td> 3天 </td&gt; <td> 1:10（1 + 9） </td><td> 1:20（1 + 19） </td></tr><tr><td colspan="3"> 每ml细胞的典型数 </td></tr><tr><td></td><td> MDCK细胞 </td><td> MDCK-SIAT1细胞 </td></tr><tr><td> 2天 </td><td> 2×10 5个 </td><td> 1×10 5个 </td></tr><tr><td> 3天 </td><td> 1×10 5个 </td><td> 5×10 4 </td></tr></tbody></table> 表1:MDCK或MDCK-SIAT1细胞融合瓶的亚文化的典型稀释。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>模式</td><td>专业模式</td></tr><tr><td>文件类型</td><td>膜（该片区指南） </td></tr><tr><td>自动曝光类型</td><td>照片</td></tr><tr><td> IM年龄类型</td><td> 24位彩色</td></tr><tr><td>解析度</td><td> 1200 DPI </td></tr><tr><td>保存的图像格式</td><td> TIFF </td></tr></tbody></table> 表2:平板扫描仪的典型设置。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 行 </td><td> 病毒 </td><td> 建议稀释 </td></tr><tr><td> 一个 </td><td> A /卡梅伦/ 15V-二千〇一十六分之三千五百三十八</td><td> 1.0×10 -2 </td></tr><tr><td> 乙 </td><td> A /符拉迪沃斯托克/2015分之36 </td><td> 1.0×10 -3 </td></tr><tr><td> C </td><td> A /莫斯科/二千〇一十五分之一百〇三</td><td> 1.1×10 -1 </td></tr><tr><td> ð </td><td> A /托木斯克/二千〇一十五分之五</td><td> 5.9×10 -1 </td></tr><tr><td> Ë </td><td> A /莫斯科/二千〇一十五分之一百〇一</td><td> 8.3×10 -1 </td></tr><tr><td> F </td><td> A /莫斯科/二千零十五​​分之百</td><td> 1.4×10 -2 </td></tr><tr><td> G </td><td> A /莫斯科/二千〇一十五分之一百三十三</td><td> 1.3×10 -2 </td></tr><tr><td> H </td><td> A /布拉迪斯拉发/二千〇一十五分之四百三十七</td><td> 1.3×10 -4 </td></tr></tbody></table> 表3:从30％ICP的人口计算的病毒稀释液。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 柱 </td><td> 抗血清 </td><td> 推荐滴度 </td></tr><tr><td> 1 - 2 </td><td> A / HK4801 / 2014 </td><td> 110 </td></tr><tr><td> 3 - 4 </td><td> A / HK7295 / 2014 </td><td> 213.3 </td></tr><tr><td&gt; 5 - 6 </td><td> A /南非/ R2665 / 2015 </td><td> 2155.8 </td></tr><tr><td> 7 - 8 </td><td> A /瑞士/二千〇一十三分之九百七十一万五千二百九十三</td><td> 89.4 </td></tr><tr><td> 9 - 10 </td><td> A /荷兰/二千〇一十四分之五百二十五</td><td> 78.6 </td></tr></tbody></table> 表4:A /斯德哥尔摩的50％ICP /二千零十五分之六十三反对抗血清滴度。

Watch this Scientific Journal Video about Optimization of a Quantitative Micro-neutralization Assay at JoVE.com

Optimization of a Quantitative Micro-neutralization Assay

这项研究描述了一个 成像基于微中和测定来分析病毒之间的抗原关系。该协议采用平板扫描仪，有四个步骤，包括滴定，滴定定量，中和，中和定量。该法与目前流行的流感A（H1N1）pdm09，A（H3N2）和乙型流感病毒效果很好。

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

optimization-of-a-quantitative-micro-neutralization-assay

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

20.9K 次观看.  The Francis Crick Institute. 这项研究描述了一个 成像基于微中和测定来分析病毒之间的抗原关系。该协议采用平板扫描仪，有四个步骤，包括滴定，滴定定量，中和，中和定量。该法与目前流行的流感A（H1N1）pdm09，A（H3N2）和乙型流感病毒效果很好。 

视频: Optimization of a Quantitative Micro-neutralization Assay

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

酶标菜生物膜形成实验

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

科研

免疫与感染

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

活体显微镜的脾:寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

细胞迁移的的Phagokinetic轨道运动MLCK的定量评价

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

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 ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

人中性粒细胞迁移的跨越培养膀胱上皮定量评价

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...