该项研究工作得到由谭医院个人种子资助计划2015中心资助;新加坡眼科研究所的试点补助金和谭医院的资金补助。

本研究中使用的协议已获新加坡谭家医院机构审查委员会批准. 
 1. 撕裂细胞因子分析 <ol> <li> 催泪集: <ol> <li> 要求受试者舒适地坐在检查椅上, 并将其头部置于头枕上. </li>
		<li> 请主题查找, 仔细打开斯戈默条 (由惠特曼滤纸41制成), 并将条的圆端放在眼睛的下穹上, 并指示主体闭上眼睛5分钟。在收集眼泪时, 要非常小心, 尽量减少眼睛表面接触。 30 </li> <li> 要求主题打开他/她的眼睛, 删除该条并将其放置在无菌1.5 毫升离心管中。立即将导管转移到实验室或存储在-80 o C, 直到测试细胞因子分析. </li>
	</ol> </li> <li> 撕裂流条的撕裂试样的洗脱: <ol> <li> 将撕裂流条切至长度为0.5 厘米 (以规范要测试的泪水的数量) 并将其置于无菌1.5 毫升离心管中. </li>
		<li> 添加30和 #181; 检测缓冲液, 室温孵育5分钟, 然后在 1.4万 x g 处离心, 1 分钟. </li>
		<li> 将上清液转移到另1.5 毫升离心管中, 然后丢弃该条。将含有该管的样品放在冰上, 并立即使用洗撕裂样品进行细胞因子分析. </li>
	</ol> </li> <li> 准备试剂: 注: 在每个样品中测试的四十一分析, 采用珠基复合法测定, 分别为白细胞介素 (il)-1-#945;、IL-1 和 #946;、IL 和 #945;、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-12 p40, IL-12 p70, IL-13, IL-15, IL-17A, 干扰素-α (干扰素-#945;) 2, 干扰素和 #947;, 干扰素-伽马诱导蛋白 10 (IP-10, CXCL10), 巨噬细胞衍生的趋化因子 (MDC), 巨噬细胞炎症蛋白 (MIP)-1 和 #945; 和 MIP-1 和 #946;, 单核趋蛋白 (MCP)-1, MCP-3, 肿瘤坏死因子α (tnf-和 #945;), tnf-和 #946; 生长调节的癌基因, 肿瘤生长因子α (TGF #945;), 血管内皮生长因子 (VEGF), 表皮生长因子 (EGF), 成纤维细胞生长因子-2, 血小板衍生生长因子-AA, 增殖素-AB/BB, 粒细胞集落刺激因子 (g-csf), 粒细胞-巨噬群刺激因子 (GM-csf), 细胞, fractalkine, 可溶性 CD40 配体 (sCD40L), Fms 样酪氨酸激酶3配体 (Flt-3L), 调节激活, 正常 T 细胞表达和分泌蛋白 (RANTES)。
	<ol> <li> 将抗体珠溶液 (50X) 瓶带到室温 (20 和 #160;-25 o C), 并将小瓶旋转1分钟。
		<li> 统计化验所需的水井数, 并计算该化验所需的总抗体珠鸡尾酒溶液 (25 和 #181; L) 和每个抗体珠溶液 (50X) 的数量. </li>
		<li> 通过添加每个抗体珠溶液的计算量来准备鸡尾酒解决方案, 并通过加入剩余的珠稀释剂填充到所需的最终体积。确保鸡尾酒中每个抗体的最终浓度是1X。总是准备至少20% 额外量的鸡尾酒解决方案, 以防移错误. 
		注: 作为一个例子, 为96井化验准备3000和 #181; 抗体珠鸡尾酒解决方案的 L。每个抗体珠溶液所需的量 = 3000/每种抗体珠溶液的浓度;3000/50 = 60 和 #181; 每个抗体珠解决方案的 L <ol> <li> 添加60和 #181; l 每41抗体珠解决方案 (60 X 41 = 2460 和 #181; l) 成鸡尾酒瓶, 并添加540和 #181; 珠稀释液对抗体珠合剂进行3000和 #181; 最终的 l工作解决方案 (1X). </li>
			<li> 使用前, 适当混合珠鸡尾酒解决方案。在检测后, 将剩余的卷存储在 2-8 o C 上, 最多30天. </li>
		</ol> </li> <li> 通过添加250和 #181 来准备质量控制 (qc); 去离子水到 qc 1 和 qc 2 库存. </li>
		<li> 通过将瓶子反转数次和十年代的漩涡来正确混合. 将解决方案转移到正确标记的聚丙烯离心管, 并将余下的解决方案存储在-20 和 #176; C 可用于最多30天. </li>
		<li> 通过将270毫升去离子水添加到30毫升的10X 洗涤缓冲液中, 并混合好 (稀释前, 将10X 缓冲带到室温, 并通过混合溶解所有的盐沉淀物) 来准备洗涤缓冲器。将未使用的洗涤缓冲器 (1X) 存储在2-8 和 #176; C 可用于最多30天. </li>
		<li> 通过添加250和 #181 来准备人体细胞因子标准库存 (所有分析的 1万 pg/毫升); 去离子水到储存瓶混合好通过多次倒置和漩涡小瓶十年代. 允许它站立10分钟, 并将库存解决方案转移到正确标记的聚丙烯离心管。化验后, 将余下的溶液储存在-20 及 #176; C, 可用于30天. </li>
		<li> 使用5倍的串行稀释 (50 和 #181; l 和 #62; 200 和 #181; l) 用化验缓冲器来准备工作人体细胞因子标准, 以获得2000、400、80、16和 3.2 pg/毫升. </li>
		<li> 标准准备后, 使用60分钟内的工作标准, 并使用化验缓冲作为空白/背景 (-pg/毫升). </li>
	</ol> </li> <li> 基于多路检测的细胞因子分析: <ol> <li> 将所有试剂置于室温和漩涡中5-10 秒后再加入到96井孔板中。如果洗撕裂样品被储存在-80 o C 之前的化验, 解冻冰冻撕裂提取物的冰和离心机在 1000 X g 5 分钟 </li>
		<li> 为工作人体细胞因子标准 (0 (空白)、3.2、16、80、400、2000和 1万 pg/mL)、QC1、QC2 和样品准备一个垂直配置的化验工作表. </li>
		<li> 添加200和 #181; L 1X 洗涤缓冲器的每一个良好的板, 密封它与板封口机, 并保持它在一个平板振动筛室温 (20-25 o C) 为 10 min. </li>
		<li> 醒酒1X 洗涤缓冲器通过倒置板, 并点击它到吸水毛巾几次, 以消除任何残留量的洗涤缓冲区在水井. </li>
		<li> 添加25和 #181; 每个工作的人体细胞因子标准, QC1, QC2, 空白 (化验缓冲) 和样本到适当的井. </li>
		<li> 添加25和 #181; 对每个井进行化验缓冲. </li>
		<li> 在每个井中添加25和 #181; 1X 抗体珠鸡尾酒溶液。由于抗体珠溶液是轻敏感的, 密封板与板封口机和覆盖它与铝箔, 以保护它从光期间的化验. </li>
		<li> 将盘在 4 o C 在摇床上过夜. </li>
		<li> 将板放在自动磁板垫圈的板架上。让它坐1分钟, 以解决在底部的磁性珠子和吸井的内容。添加200和 #181, 每井洗涤缓冲器, 并让它坐1分钟, 然后抽吸井的内容。再重复一次洗涤 (为洗盘子, 跟随成套制造商和 #39; s 指示). </li>
		<li> 添加25和 #181; 在每个井中检测抗体溶液, 密封板, 用铝箔覆盖, 并在室温下孵育60分钟的振动筛. </li>
		<li> 添加25和 #181; Streptavidi藻溶液放入每个井中, 密封板, 用铝箔覆盖, 并在室温下孵育30分钟的振动筛. </li>
		<li> 将板放在磁板垫圈上。让它坐1分钟, 然后抽吸好的内容。添加200和 #181, 每井洗涤缓冲器。让它坐1分钟, 然后抽吸好的内容。重复一次洗涤. </li>
		<li> 添加150和 #181; 每个井的鞘液 L, 并放置在一个振动筛5分钟的室温, 以重抗体珠的板. </li>
		<li> 使用基于磁珠的多重检测板读取器立即读取车牌, 并使用5参数曲线拟合算法分析细胞因子的浓度。在 图 1 中显示了撕裂细胞因子分析的示意图流程图. </li>
	</ol> </li> <li> 读取检测板 (仪器设置) 和数据分析: <ol> 在基于磁路的多路检测读取器上切换, 并预热激光30分钟. <li>
		<li> 启动基于磁珠的多重检测软件。根据和 #39; 自动维护和 #39; 选项卡, 选择和 #39; 校准-验证和 #39; 选项。涡旋每个试剂瓶的校准和验证珠三十年代. 将每种试剂的5滴放入指定的井中。用去离子水和70% 乙醇填充指定的水库. </li>
		<li> 创建新协议 <ol> <li> 打开和 #39;P rotocols 和 #39; 页然后 #39;P rotocols 和 #39; 选项卡. 单击和 #34; 创建新的协议和 #34;; #39; 设置和 #39; 选项卡将打开 </li>
			<li> 在 #39; 名称和 #39; 框中, 键入协议的名称. </li>
			<li> 在 #39 的右侧框中键入说明; 名称和 #39; 框. </li>
			<li> 在 #39; 版本和 #39; 框中, 键入协议的版本. </li>
			<li> 在 #39; 制造商和 #39; 框中, 键入协议的制造商信息. </li>
			<li> 定义和 #39 中的设置; 购置设置和 #39; 部分, 如下所示。设置和 #39; 容量和 #39;: 100 和 #181; #39; 超时和 #39;: 六十年代;#39;DD 门 #39;: 8000 到 1.5万;#39; 记者增益和 #39;: 标准 PMT;#39; 珠型和 #39;: 磁珠. </li>
			<li> 定义和 #39 中的设置; 分析设置和 #39; 部分, 选择和 #39; 定量和 #39; 作为分析类型。设置和 #39; 标准和 #39 的数量;: 6;#39; 控制和 #39 的数量;: 0;#39; 复制和 #39 的平均值; 框; 
			在获得样品和 #39 时, #39; 分析结果; 框. </li>
			<li> 单击和 #34; 下一 #34;; #39; 分析和 #39; 选项卡打开, 单击所需的分析 (珠子 ID) 在已编号的分析物网格中. </li>
			<li> 在 and #39 中单击并键入相应的分析物名称; 名称和 #39; 分析物网格右侧的列 (分析和 #39; 名称及其相应的磁珠区域详细信息在 表 1 ) 中提到. </li>
			<li> 单击并键入所需的度量单位 (即 pg/mL) 在和 #39; 单位和 #39; #39 的左侧框; 应用所有和 #39; 按钮。然后单击并 #34; 应用所有 #34;. </li>
			<li> 在 #39 中单击并键入每个分析物 ( 即 50) 所需的磁珠计数; 计数和 #39; 框。单击并 #34; 应用所有 #34;. </li>
			<li> 单击并 #34; 下一 #34;; "板布局" 选项卡打开。为标准和选择 #34; 2 和 #34; 在 "复制计数" 下, 单击 "与 #34; S 和 #34; 标准按钮。为背景和 #34 重复此步骤; B 和 #34; 以及样品和 #34; U 和 #34; 井. </li>
			<li> 单击并 #34; 保存和 #34;. </li>
		</ol> </li> <li> 创建新的标准/控制批次 <ol> <li> 打开和 #39;P rotocols 和 #39; 页, 然后 #39; 标准和 #38; 控件和 #39; 选项卡. 单击和 #34; 创建新的标准/控制批次和 #34;... </li>
			<li> 打开和 #39; 选择协议和 #39; 框。选择在步骤1.5.3 中创建的协议. </li>
			<li> 相应标准信息的关键字: std/ctrl 套件号、std/ctrl 套件名称、过期和制造商. </li>
			<li> 每个分析物的最高标准值的键 ( 即 10000 pg/毫升)。在5的关键在稀释和点击和 #34; 应用所有 #34; 自动生成其余标准的预期浓度. </li>
			<li> 单击并 #34; 保存和 #34;. </li>
		</ol> </li> <li> 从现有协议中创建新批处理 <ol> <li> 打开和 #39; 批次和 #39; 页并单击和 #34; 从现有协议和 #34 创建新的批处理选项卡.. </li>
			<li> 在 "和 #39 中键入批次名称; 批次名称和 #39; 框, 然后在" 和 "#39 中键入有关批次的说明; 输入可选说明和 #39; 框. </li>
			<li> 单击先前生成的协议 (在步骤1.5.3 中). </li>
			<li> 单击和 #34; 下一页 #34;; 下一个选项卡是 #39; 性病和 #38; Ctrls 和 #39; 选项卡. 查看检测标准的详细信息, 选择和 #34; 下一页和 #34;. </li>
			<li> 在 #39;P 后期布局和 #39; 选项卡上, 为该批分配良好的命令, 然后单击并 #34; 保存和 #34; 将批处理信息保存到 #39;P 结束批次和 #39; 列表. </li>
			<li> 将该板加载到基于磁路的多路检测板读取器中, 摇动它5分钟, 然后从待定的批处理列表中运行批处理。获取的数据将以. csv 文件格式保存. </li>
		</ol> </li> </ol> </li> <li> 数据分析 <ol> <li> 使用 rbx 转换软件将从基于珠子的多路检测软件生成的. csv 文件转换为. rbx 文件 (结果数据文件) 格式。启动转换软件, 选择 xPONENT 文件下的. csv 文件, 然后单击 #34; 生成和 #34; 按钮;. rbx 文件将保存在选定的输出文件夹中. </li>
		<li> 启动分析软件并打开. rbx 文件. </li>
		<li> 使用 4 pl/5 pl 曲线拟合优化标准曲线。
		<ol> <li> 在和 #39 中选择以下内容; 标准曲线和 #39; 选项卡: 和 #39; 回归类型和 #39;: #39; Logistic-5PL 和 #39;; #39; 轴变换和 #39;; #39; 日志 (x)-线性 (y) 和 #39;; 勾选和 #39; 显示具体范围线和 #39; 框; 勾选 #39; 显示未知样品和 #39; 箱;勾选和 #39; 显示控制样本和 #39; 框;勾选 #39; 在所有的分析和 #39 中应用;勾选 #39; 在优化和 #39 后显示报告; 框. </li>
			<li> 单击 #34; 优化和 #34; auto-optimization 按钮. </li>
		</ol> </li> <li> 在 #39 下标记示例标识; 输入示例信息和 #39; 选项卡. </li>
		<li> 获取 #39 中的观察到的浓度; 报表表和 #39; 选项卡, 然后单击并 #34; 导出报表表和 #34; 在电子表格文件中生成数据以进行进一步分析. </li>
	</ol>
	</li>
</ol>

作者没有什么可透露的。没有经济利益或利益冲突。

细胞因子是小细胞分泌的蛋白质和有效的免疫调节调控免疫应答。32撕裂标本中各种细胞因子的表达谱与眼睛的各种病理条件有关, 研究细胞因子图谱有助于了解疾病发病机制, 确定眼部健康状况,疾病的严重性, 诊断和进展。2,5较低的蛋白质浓度和小样本量是传统方法的主要挑战, 限制了对撕裂细胞因子的分析, 如 ELISA 法的使用。基于<su...

眼泪是由泪腺和副腺体产生的, 并覆盖眼睛的外表面。泪膜由外脂层和内水层组成, 包括可溶性蛋白、粘和膜结合粘。眼泪可以防止微生物侵入, 提供营养, 并为眼部表面润滑。眼泪作为空气和组织之间的接口, 用于氧气输送到角膜。1撕裂膜由蛋白质、碳水化合物、血脂和电解质组成。一些研究发现, 泪液蛋白与眼部和全身性疾病如青光眼、干眼病、春季结膜炎、糖尿病、甲状腺相关眼眶和癌症之间的关联。2,3,4撕裂样品可以通过 microcapillary 管或撕裂流条 (斯戈默条) 收集。此外, 斯戈默的测试是在角膜和屈光手术诊所中执行的标准程序, 其结果可用于细胞因子分析化验。无创样本采集, 生物标本的可及性, 撕裂成分与各种生理和病理条件的联系, 使泪膜成为几种眼部和全身性疾病的生物标志物的潜在来源。4,5,6
泪液细胞因子在研究各种眼病的眼表健康和炎症状态方面起着重要的作用。7撕裂标本中几种细胞因子的异常浓度报告与干眼病、春季结膜炎 (VKC)、特应性结膜炎 (AKC)、季节性过敏性结膜炎和葡萄膜炎有关。8,9,10,11,12,13撕裂蛋白可以用质谱、印迹和酶联免疫吸附法等传统方法进行分析。14,15然而, 这些方法的局限性是敏感性差和大量的样本需要分析的多个撕裂细胞因子在每个病人。16,以17微珠为基础的复配分析方法, 对复杂混合样品中的多分析进行了研究, 成功地应用于撕裂样品, 分析了不同疾病中的多种细胞因子。6,18结合了三明治 elisa 和流式细胞术技术, 使得这些检测方法比 elisa 更灵敏, 可以对单个样本中的多个分析进行量化。19该方法可应用于多种临床标本和细胞培养清, 有助于研究几种病理生理条件下的免疫应答。20,21,22,23,24
有几项研究比较了珠基多重检测与 ELISA 法的相关性, 并报告了这些方法之间的相互关系。厕所et al.比较了基于微珠的多重检测与常规 ELISA 法测定人血清或血浆样品中脂联素、抵抗素、瘦蛋白和生长激素的含量, 并报告了两者之间的强相关性 (r 和 #62; 0.9)。25杜邦et al.报告了在 IL-6 和脂多糖刺激全血的 IL-1β、IL-4、IL-5、IL-10、节能、干扰素-植物和 TNF-α的检测中, 珠基复合测定与 ELISA 之间的强相关性。从怀孕妇女收集。26平克林et al.报告了另一种强关联度检测血清抗体对乙型流感嗜血杆菌(r = 0.96), 毒素的测定破伤风梭菌 (r = 0.96) 和杆菌白喉(r = 0.91)。27 Biagini et al.报告了一个高正相关 (r = 0.852) 之间的珠基复合测定和 ELISA 检测的芽孢杆菌抗体在血清样品中的抗 PA IgG。28 Wang et al.报告了在检测阿尔茨海默病生物标志物淀粉样蛋白β 42 (r = 0.77), 总 tau (r = 0.94), 和头磷酸化的氨基酸 181 (r = 0.82) 之间的相关性。脑脊液样本29这些研究证明了基于微珠的多重分析方法对临床样品的多样性、小样本量的要求以及与标准 ELISA 的相关性, 使微珠基复合检测成为一个有前途替代传统的 ELISA 方法检测不同类型的样品中的多种分析的疾病表型。在这里, 我们描述了一个标准化的协议, 以珠子为基础的多重检测细胞因子分析41分析的撕裂样本从健康的主题使用斯戈默带。

<table>
	<tbody>
		<tr>
			<td>Milliplex MAP human cytokine / chemokine magnetic bead panel -1 kit</td>
			<td>Merck, USA</td>
			<td>HCYTOMAG-60K-41</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexmap 3D luminex instrument&#160;</td>
			<td>Luminex Corp, Austin, TX, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>xPonent software&#160;</td>
			<td>Luminex Corp, Austin, TX, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RBXGenerator software</td>
			<td>BIO-RAD, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Plex Manager 6.1</td>
			<td>BIO-RAD, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plate shaker</td>
			<td>Corning, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TECAN Microplate Washer</td>
			<td>Tecan, Switzerland</td>
			<td>HydroSpeed</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 2</td>
			<td>Scientific Industries inc, USA</td>
			<td>G560E</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Mettler Toledo, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml microcentrifuge tube</td>
			<td>Axygen, USA</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Schirmer tear flow test strip</td>
			<td>Eye Care and Cure, USA</td>
			<td>101657</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexmap 3D Calibration Kit</td>
			<td>Luminex Corp, Austin, TX, USA</td>
			<td>40-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexmap 3D Verfication Kit</td>
			<td>Luminex Corp, Austin, TX, USA</td>
			<td>40-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular examination chair</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

bead based multiplex assay for analysis of tear cytokine profiles

泪膜是一种复杂的脂质、蛋白质和矿物质的混合物, 覆盖了眼睛的外表面, 从而为底层细胞提供润滑、营养和保护。泪流分析是鉴别各种眼部疾病的预测、诊断和预后的标志物的一个新兴领域。眼泪是容易接近, 他们的收集是无创的。因此, 在研究蛋白质或代谢物组成的变化及其与病理条件的关系时, 先进的技术正日益突出, 以识别多分析的泪水。泪液细胞因子是研究眼部健康状况的理想标志物, 也有助于了解眼部疾病和春季结膜炎等不同眼表疾病的发病机制。基于微珠的复用检测具有较高的灵敏度, 能在少量样品中探测多个分析。在这里, 我们描述了一个标准的催泪样本收集, 提取和分析细胞因子的特征, 采用微珠的多重检测。

用泪流条和细胞因子水平分析了8眼4健康受试者的泪液样本。所有科目均为男性, 四受试者的年龄分别为36、42、44及52岁。通过临床试验 (泪膜破裂时间、斯戈默试验、角膜染色、结膜染色、眼睑/板腺检查、角膜撕裂征象和视觉体征及症状), 对眼部健康和干眼病进行评估。国际干眼研讨会, 2007 指导方针, 没有一个主题显示任何迹象和症状的干眼病。31
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Watch this Scientific Journal Video about Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles at JoVE.com

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

泪膜细胞因子的分析有助于研究各种眼部疾病。基于微珠的复合检测方法简单、灵敏, 能够对小体积样品中的多个目标进行测试。在这里, 我们描述了一个协议的撕裂膜细胞因子分析一个使用珠基复用法。

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 ...

bead-based-multiplex-assay-for-analysis-of-tear-cytokine-profiles

Research

JoVE Journal

Immunology and Infection

11.7K 次观看.  Tan Tock Seng Hospital. 泪膜细胞因子的分析有助于研究各种眼部疾病。基于微珠的复合检测方法简单、灵敏, 能够对小体积样品中的多个目标进行测试。在这里, 我们描述了一个协议的撕裂膜细胞因子分析一个使用珠基复用法。

视频: Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

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Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

科研

免疫与感染

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 ...

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 ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

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 ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

人外周血 NK 细胞活性评估流基于流式细胞仪检测细胞毒性检测

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

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

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches.
Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay.
In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

This protocol describes the quantification of multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using multiplex bead based immunoassay platform and a flow cytometer.

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 ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

动态粘附法在炎症性肠病抗粘连治疗功能分析中的作用

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...