Name: counting proteins in single cells with addressable droplet microarrays
Uploaded: 2018-07-06T13:00:00.000+00:00
Duration: 12 min 25 s
Description: Watch this Scientific Journal Video about Counting Proteins in Single Cells with Addressable Droplet Microarrays at JoVE.com

ASR 设计的实验, 开发的协议和分析数据。SC 和 PS 进行了细胞大小实验。ASR 和 OC 写了手稿。提交人希望感谢 David 斯科特·克鲁格教授提供设备的支持。作者希望感谢帝国学院先进的 Hackspace, 以获得制造和原型设施。

1. 准备
<ol><li>
制作芯片和打印抗体微阵列
	<ol>
<li>将粘合剂硅胶/丙烯酸隔离器附着在盖玻片功能上, 以支持抗体芯片。这被称为芯片。 注: 各种表面化学试剂已经过测试, 以适合与可寻址的水滴。5表面化学物质可能需要对替代捕获剂进行优化。ADM 隔离器是可利用的商业或可由激光切割丙烯酸 (CAD 文件为隔离器使用的这项工作是作为下载提供; 请参阅补充代码文件)。</li>
		<li>打开 microarrayer, 将湿度设置为75%。 注意: 相对湿度减少了芯片针的打印溶液蒸发, 减少了内部和点间的变化。</li>
		<li>用超声波清洗针清洗液中的微阵列针5分钟。用洗瓶器用超纯水冲洗别针, 用氮气干燥。 注: 将 pin 挂起, 只浸入针脚尖端。如果无法获得 pin 夹, 则用适当钻孔的一组孔进行显微镜滑动会有帮助。</li>
		<li>制作5毫升的打印缓冲器, 包括 3 x 盐水钠 (SSC) 缓冲器, 1.5 米甜菜碱补充0.01% 的月桂酸钠 (SDS)。储存在4摄氏度无限期。</li>
		<li>要准备一个打印解决方案, 解冻 anti-p53 抗体 (p53/Mdm2 ELISA 试剂盒; 见材料表) 整除数存储在-80 摄氏度。将其与打印缓冲器混合 1:1, 最终浓度为0.5 毫克/毫升。使用微和 microarrayer 中的位置在384µL 中加载5-10 的打印解决方案。</li>
		<li>加载芯片组装在步骤1.1.1 进入 microarrayer。程序的 microarrayer 打印点的坐标定义的每个井的隔离器。 注: Microarrayers 采用了一系列的、主要的专有软件, 因此鼓励读者查阅相关文献。在1.1.1 中, 伴随 CAD 文件中的 ADM 隔离器所需的微阵列包括矩形元件;提供了相关的距离。</li>
		<li>将芯片存放在密闭的容器中, 用箔包装。存储在4°c 6 周。 注: 箔是为了防止任何照片损坏。存储在4°c 限制生物分子的降解率和任何可能采取行动减少微阵列活动的有害反应。密闭容器允许芯片在使用前平衡到室温, 而不会在芯片表面形成冷凝。接触芯片印刷利用表面张力和附着力之间的打印解决方案和印刷承印物产生斑点。印前或印迹通常需要从微阵列中去除多余的溶液, 以产生均匀大小的斑点。不要丢弃牺牲预打印盖玻片, 因为它可以用来评估批次质量。</li>
	</ol>
</li>
	<li>
准备注射器, 油管和同心喷嘴来分配可寻址的水滴
	<ol>
<li>拆卸100µL (为水滴) 和1毫升 (用于封盖油) 玻璃哈密尔顿注射器和漂洗的零件与蒸馏的 H2O。</li>
		<li>将100毫米长度的150µm ID/360 µm 外径熔融硅胶油管通过40毫米长度1.0 毫米 ID/1/16 "外径粉煤灰油管, 直到它突出了2毫米。这将形成同心喷嘴。</li>
		<li>将一层薄薄的氰胶涂在10µL 吸管尖端的末端, 并插入40毫米的1.0 毫米 ID/1/16 "外径的粉煤灰导管"。如果需要, 重新定位熔融硅胶油管, 以保持在喷嘴前的2毫米突出的粘合剂集。</li>
		<li>将熔融二氧化硅毛细管的另一端插入200毫米长 0.014 "ID/0.062" 外径聚四氟乙烯油管的一端, 并将其连接至100µL 哈密尔顿注射器, 填充有4% 牛血清蛋白 (BSA) 的磷酸盐缓冲盐水 (PBS) (PBSA)。 注意: 蛋白质和其他生化物种可能非特异地绑定到表面, 并且可以丢失或变性。BSA 用于 "阻止" 曲面, 通过牺牲绑定到这些曲面来最小化非特定绑定。</li>
		<li>插入一个400毫米长1.0 毫米的 ID/2.0 毫米外径 FEP 油管到200µL 吸管提示, 直到它形成一个印章。将一层薄薄的氰胶水涂到另一200µL 吸管尖端, 并将其推入第一个尖端, 以固定油管到位。将 1.0 mm ID/2.0 mm 外径 FEP 油管的开口端连接到一个1毫升的哈密尔顿注射器中, 填充矿物油。</li>
		<li>将200µL 吸管尖端组件插入同心喷嘴的10µL 吸管尖端。将注射器放在单独的注射器泵中, 因为它们需要独立操作。</li>
		<li>用 4% PBSA 阻断溶液填充100µL 注射器。用堵塞溶液重新连接并冲洗 "水" 油管。重复两次, 总共3个刷新。</li>
		<li>填充100µL 注射器与0.125 µg mL-1检测抗体 (anti-p53 抗体 (DO-1) 标签与 Alexa 488) 在 4% PBSA 和重新连接油管。用25µL 检测抗体溶液代替油管中的堵塞液。</li>
		<li>用矿物油冲洗 "油" 油管, 直到所有油管和喷嘴充满油。用矿物油重装1毫升注射器, 再附加油管。将油管和喷嘴组件固定到 XYZ 机械手上。</li>
	</ol>
</li>
	<li>
准备 microinjector 和机器人 注: 下面的步骤具体提到材料表中指定的 microinjector 和机器人设备的组件, 但通常适用于任何此类设备。<ol>
<li>通过附加压力管和毛细管支架装配 microinjector。</li>
		<li>慢慢旋转活塞表盘, 直到矿物油完全填满线。</li>
		<li>将毛细管支架装入平移头安装。</li>
		<li>用握头固定毛细管的毛细管。</li>
		<li>将 4% PBSA 的溶液注入隔离器的一个井中。</li>
		<li>使用机器人进行翻译, 并将毛细管的尖端浸入溶液中。</li>
		<li>慢慢地用 4% PBSA 填充毛细管, 然后离开以阻挡10分钟。</li>
		<li>弹出 microcapillary 并旋转平移模块, 使组件清除芯片。</li>
	</ol>
</li>
</ol>2. 用单细胞形成可寻址的水滴和负载
<ol><li>形成可寻址的水滴
	<ol>
<li>在显微镜阶段保护芯片。 注: 显微镜是一种倒置荧光自动显微镜, 可单分子 TIRF 装有一个编码 XY 阶段和电子倍增电荷耦合装置摄像头 (EM CCD)。</li>
		<li>使用自动显微镜控制软件记录阵列中每个点的显微镜阶段坐标。</li>
		<li>在显微镜阶段设置 XYZ 机械手。把喷嘴放在50-60°的角度。确保喷嘴有足够的长度和间隙到达芯片。设置 ' 水 ' 和 ' 油 ' 注射器泵分配 10 nL 在100µL/分钟和5µL 在100µL/分钟, 分别。 注: 在准备过程中, 喷嘴头部的水溶液可能会干燥。</li>
		<li>分配水溶液直到喷嘴头部可见液体珠子。用无尘擦拭擦除。 注: 根据调查的条件, 预留数口水井作为细胞的储层。对于单个单元格线/类型单个水库就足够了。</li>
		<li>使用自动显微镜控制软件, 将显微镜阶段坐标设置为阵列中的抗体点, 并将焦点放在盖玻片表面。</li>
		<li>小心不要扰乱现场, 使用 XYZ 机械手, 将同心喷嘴的玻璃毛细管尖端对准抗体点的一侧, 并分配 10 nL 水溶液。没有移动的阶段, 分配5µL 的油来盖水溶液。慢慢地提高喷嘴清除液滴和移动到下一个井。</li>
		<li>对阵列中的所有抗体点重复步骤2.1.6。</li>
		<li>30分钟后, 图像所有斑点在阵列使用自动显微镜控制软件, 以确定单一分子的背景, 每一个抗体斑点之前, 加载细胞。</li>
	</ol></li>
	<li>用单元格加载可寻址的水滴
	<ol>
<li>使用胰蛋白酶/EDTA 溶液从孵化器中取出细胞培养瓶和分离细胞。 注: 本研究采用 Dulbecco 修饰鹰培养基 (DMEM) 对人结肠癌细胞系进行培养, 并辅以 10% (v/v) 胎牛血清 (血清), 共2个孵化器。</li>
		<li>如果需要, 荧光染色细胞。<ol>
<li>并用重悬细胞中的0.125 微克/毫升检测抗体 (anti-p53 抗体 (DO-1) 标签与 Alexa 488) 10% 血清在 L-15 媒体。为了确保单个细胞悬浮, 过滤细胞溶液通过40µm 间距细胞过滤器。</li>
		</ol>
</li>
		<li>用 hemocytometer 计数细胞, 稀释或浓缩细胞溶液浓度为 25-400 x 103细胞/毫升。这确保了细胞沉积有足够的间距, 以舒适地操纵 microcapillary。</li>
		<li>使用机器人和 microcapillary 将单个细胞从细胞库中加载到可寻址的水滴中。 注意: 非粘附细胞可以大量加载到微中, 并将一个人分成一个可寻址的水滴。尽管表面阻断处理, 黏附细胞线将倾向于不明确地坚持玻璃 microcapillary 内壁和丢失。</li>
		<li>使用10×目的观察, 使用 microinjector 从细胞库中吸入单个细胞进入 microcapillary。</li>
		<li>如果使用电子机械手舞台或手动记下 z 位置, 则存储机器人阶段坐标。</li>
		<li>将微向上平移, 以清除芯片的1毫米高度。用操纵杆手动执行此操作, 或自动使用电子机械手阶段的 "弹出" 功能。</li>
		<li>使用自动显微镜控制软件将舞台坐标设置为可寻址的水滴。' 注入 ' 微通过返回到存储 (或指出) z 位置。使用操纵杆手动执行此操作, 或在采用电子机械手阶段时自动进行。 注: microcapillary 将刺穿盖油并且位于可寻址的水滴的水部分之内。</li>
		<li>使用 microinjector 在可寻址的水滴中分配单元格。10 nL 寻址液滴的体积将增加不到1%。</li>
		<li>重复 2.2. 5-2. 2.9 为剩余的可寻址的水滴留下一些免费的实验控制, 其中可寻址的水滴不包含细胞。 注意: 使用 microcapillary 和机器人将单个单元格加载到可寻址的水滴的一个简单的替代方法是在步骤1.2.8 中替换该解决方案, 并在 4% PBSA 中包含细胞的检测抗体的解决方案, 其浓度为105细胞/毫升, 相当于 1 cell/10 nL。单细胞占用将泊松, 细胞浓度将需要优化。</li>
	</ol></li>
	<li>溶解细胞和图像阵列
	<ol>
<li>brightfield 显微镜下的水滴中的图像细胞, 包括任何荧光成像。 注: 成像需要大约3分钟的图像 ~ 100 滴使用自动显微镜。用 60 NA = 1.49 的油浸泡目标进行成像。brightfield 成像不需要过滤器, 而标准过滤器集用于荧光成像。</li>
		<li>使用单分子 TIRF 显微镜对阵列中的所有斑点进行图像处理。 注意: 设置在步骤2.3.1 中用作专用的 TIRF 筛选器集。这些图像将随后分析在3节, 以确定的背景下, 单个分子绑定到每个抗体斑点之前, 裂解。使用全内反射荧光 (TIRF) 显微镜的单分子成像大约需要5分钟到100点的图像。</li>
		<li>聚焦在一个可寻址的水滴中的细胞上。通过聚焦单个 6 ns 激光脉冲 (波长 1064 nm, 脉冲能量 14.1, 每脉冲0.3 µJ) 接近细胞的位置, 实现完整的光裂解。 注: 激光脉冲建立了一个膨胀的空化气泡, 它能剪切细胞并释放细胞成分进入水滴体积。4,31由于激光诱导裂解而产生的机械过程不会干扰低脉冲能量的油水界面。光裂解通常需要大约20-30 分钟溶解100细胞。正如讨论部分所讨论的那样, 如果不能进行光学裂解设置, 就有许多替代方法来溶解单个细胞。</li>
		<li>对所有含有细胞的可寻址液滴重复5个自由的实验控制, 其中可寻址的水滴含有不裂解的细胞。</li>
		<li>记下每个单元格裂解的时间。 注意: 这些时间将用于纠正步骤3.1.9 中每个点的单个绑定曲线。通常, 要注意第一个和最后一个单元格是裂解的, 并且估计其余的时间假设在裂解事件之间有足够的一致性。</li>
		<li>使用 TIRF 显微术获取单个分子图像, 每10分钟为前30分钟, 然后每20分钟为60分钟。如果只对蛋白质在平衡中的数量有兴趣, 在室温下孵化芯片90分钟后, 图像所有斑点。 注: 达到平衡的时间取决于液滴体积和检测中所用抗体的亲和力。TIRF 显微镜是使用激光励磁源在 = 488 毫微米和1.5 兆瓦功率测量在后光圈使用光功率计。通过将 EM CCD 摄像机的采集设置设置为900毫秒采集时间, 16 位数字化, 1 MHz 读出率和 em 增益因子 10, 获得单分子图像。隔离器可通过仔细拆卸盖玻片重新使用。</li>
	</ol></li>
</ol>3. 数据分析
<ol><li>
单分子计数 (非拥塞/数字体制)
	<ol>
<li>使用斐济 (或 Matlab), 对于任何非拥塞抗体点, 加载在裂解前获得的图像 (背景, BKD)。 注意: 使用斐济图像分析软件直接执行以下步骤中的操作。在以下链接 https://imagej.nih.gov/ij/docs/guide/146.html 中可以找到用户指南。</li>
		<li>在斐济, 选择 BKD 图像。在 "图像" 菜单中选择 "复制" 来复制 BKD。选择重复图像并在 "进程 > 筛选器" 下, 选择 "高斯模糊" 并指定50像素半径。</li>
		<li>将 BKD 图像通过模糊 BKD 图像分割, 产生 BKD_FLAT, 使 BKD 图像扁平化, 从而使励磁光源有有效的均匀强度分布。<ol>
<li>在 "过程" 中选择 "图像计算器...", 指定操作 "除法", 所需的图像, 并选择框 "创建新窗口" 和 "32 位 (浮动) 结果"。</li>
		</ol>
</li>
		<li>将图像中的每个像素减去 1 ("进程 > 数学", 选择 "减法..." 并指定值 1)。平均像素强度应为0。 注意: 由于所有像素的总和应该是 0, 并且任何剩余的偏移量可能更直接地得到补偿, 所以可以轻松地检查字段拼合和拼合的背景图像。</li>
		<li>在 BKD_FLAT 图像的4个角中, 选择50像素 x 50 像素区域。通过选择 "分析" 和 "度量" 来测量像素强度标准偏差 (σ)。 注意: 所选内容的摘要统计信息将显示在结果窗口中。这决定了非现场背景, 那里不可能有高密度的单一分子。可以使用默认菜单选择工具或运行宏代码 "makeRectangle (x、y、宽度、高度)" 手动选择要测量的区域;这将创建一个矩形选区, 其中 x 和 y 参数是左上角的坐标 (以像素为单位)。</li>
		<li>将图像阈值设置为 3σ, 并创建二进制图像 SM_MASK。<ol>
<li>在 "图像 > 调整" 下, 选择 "阈值...", 并将较低的阈值级别设置为3σ。 注意: 在阈值以下的像素被设置为零, 超过阈值的像素设置为1。阈值将决定阈值像素属于单个分子的置信度。</li>
		</ol>
</li>
		<li>在分割图像 SM_MASK 中, 通过选择 "分析" 后跟 "分析粒子" 并指定大小和圆形, 将像素强度值设置为零, 不具有4-9 像素2和圆形 0.5-1 的任何对象。 注意: 像素大小可能需要优化其他显影和显微镜设置 ups。由于相机噪声的类型, 单像素可能高于此阈值, 而不会被丢弃, 尽管不是单一分子。像素大小标准将正确丢弃此类像素。单个分子通常呈圆形。圆形值为1表示一个完美的圆圈, 而值接近0表示形状越来越长。其余的物体是单个分子, 可以计数。可以将掩码设置为标定现场的面积, 以区分每帧的现场和非现场计数。当有足够的信号当场, 然后可以应用于较早的帧时, 在以后获取的帧会更容易。</li>
		<li>对于相同的非拥塞抗体点, 加载和重复步骤 3.1.1 3.1.7 的所有图像帧捕获的时间分辨系列获得后裂解。使用步骤2.3.5 中提到的溶解时间。以更正任何绑定曲线。</li>
	</ol>
</li>
	<li>
单分子计数 (拥塞/模拟机制)
	<ol>
<li>为了计算单个分子的平均强度, 在继续之前, 重复步骤 3.1.1 3.1.8。</li>
		<li>将图像 BKD_FLAT 和 SM_MASK 相乘, 产生一个图像, 即非零像素值与单个分子相关联。<ol>
<li>要执行此操作, 请在 "进程" 菜单中选择 "图像计算器...", 指定运算 "相乘" 和所需的图像, 然后选择 "创建新窗口" 和 "32 位 (浮点) 结果" 框。</li>
		</ol>
</li>
		<li>通过选择 "分析" 和 "度量" 来求和所有像素强度值;所选内容的摘要统计信息将在结果窗口中显示。除以每步3.1.8 计数的单个分子的数量, 计算每单个分子的平均强度。</li>
		<li>对于任何拥挤的抗体点, 加载图像后溶解和扁平化的背景图像模糊的每步3.1.2 和3.1.3。</li>
		<li>将拼合图像中的每个像素减去 1 ("进程 > 数学", 选择 "减法..." 并指定值 1)</li>
		<li>在图像的4个角中选择一个50像素 x 50 像素区域, 并测量像素强度标准偏差 (σ)。有关详细信息, 请参阅步骤3.1.5。</li>
		<li>通过将图像阈值设置为 3σ, 并将像素强度值设置为小于4像素2的任何对象的零, 创建二进制图像掩码。要执行此操作, 请在 "分析" 菜单下选择 "分析粒子" 并指定大小和圆形。</li>
		<li>用二进制图像掩模将扁平拥塞抗体斑点图像相乘。<ol>
<li>要执行此操作, 请在 "进程" 菜单中选择 "图像计算器...", 指定运算 "相乘" 和所需的图像, 然后选择 "创建新窗口" 和 "32 位 (浮点) 结果" 框。</li>
		</ol>
</li>
		<li>剩余像素强度的总和, 选择 "分析" 后跟 "度量";所选内容的摘要统计信息将在结果窗口中显示。</li>
		<li>用平均单分子强度除以像素强度的总和, 以计算绑定到拥塞点的单个分子的数量。</li>
	</ol>
</li>
	<li>
绝对量化的标定曲线
	<ol>
<li>重复1和2节, 形成 10 nL 可寻址液滴与检测抗体在 4% PBSA 溶液中的已知浓度重组蛋白。</li>
		<li>通过改变添加到溶液中已知的重组蛋白浓度, 执行 102-107重组蛋白每液滴的浓度系列。建议在每液滴的蛋白质数量方面工作, 以使校准单细胞数据简单明了。</li>
		<li>使用步骤3.3.2 中的浓度序列数据来校准每一个点的任何单个分子计数, 每液滴的蛋白质丰度, 以及每单个细胞的可拓蛋白质丰度。</li>
	</ol>
</li>
</ol>

<ol>
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作者没有什么可透露的。

可寻址液滴微阵列是一种灵敏的、易于扩展的方法, 用于定量确定单个细胞内蛋白质的绝对拷贝数。
在协议中限制非特定绑定 (NSB) 的级别对于尽可能低的检测限制是至关重要的。蛋白质和其他生物化学物种可能不专门绑定到许多界面中存在的水滴-盖玻片表面, 抗体斑点和油/水界面。蛋白质可以通过分成接口或油本身而丢失。我们已经表明, 在水滴中存在的 4% BSA 可以使用协议...

该方法的目的是确定单个细胞分辨率的细胞群体中目标蛋白丰度的变化。单细胞分析提供了一些不具备传统的集成生物化学方法的好处。1,2,3,4,5首先, 在单细胞水平上工作可以捕捉到一个细胞群的丰富异质性, 否则会因传统的集成生物化学技术所产生的平均值而丧失。大多数的工作-马生物化学方法与散装工作, 要求, 因为他们经常做, 数以百万计的细胞产生的结果。当然, 评估整个细胞数量的后果取决于许多因素, 例如, 蛋白质表达的异质性可能会遗漏蛋白质丰度分布的一些重要特征。从实际的角度来看, 单细胞技术所需的灵敏度使它们能够处理大量的生物材料, 即使是更敏感的散装技术也不足以发挥作用。这方面的一个关键例子是研究稀有细胞类型, 如循环肿瘤细胞 (CTCs), 即使对于预后较差的患者, 也不到 10 CTCs 可能会出现在一个7.5 毫升的血液抽取中。6在这里, 我们提出了执行单细胞蛋白测量所需的方法, 采用减少体积抗体为基础的检测, 使用在抗体芯片上印有油的水滴。
微流控液滴平台是高通量, 能够产生数以千计的水滴每秒, 并能够隔离, 甚至培养, 单个细胞在单个的水滴, 以执行广泛的一系列生化化验。基于雾滴的技术非常适合于单细胞分析,7,8,9 , 最近的例子包括 DropSeq10和 inDrop11, 这已经大大的力量帮助放大技术。蛋白质的有限数量和不扩增方法使单细胞蛋白质组学特别具有挑战性。
液滴可通过多种方法分析, 荧光显微镜已得到广泛应用。单分子技术, 如全内反射荧光 (TIRF) 显微术, 使荧光分子能够被可视化的信噪比无与伦比。12由于消逝的领域的指数衰变, 只有显影在高接近度到表面 (顺序 100nm) 是激动的做 TIRF 一个好战略在检测少量目标分子在复杂混合物。TIRF 固有的光学切片强度也有助于避免洗涤步骤和限制化验时间和复杂性。然而, TIRF 需要平面和 TIRF 显微镜的例子, 用于流中的水滴涉及形成一个平面表面的图像。13为此, 单细胞蛋白质组技术通常以微阵列的形式在表面固定化的捕获剂周围设计微流控芯片。4,14
水滴, 本身, 可以形成在阵列上的平面表面, 所谓的水滴微阵。15,16,17在空间上组织的水滴进入阵列, 使它们能够方便地编制索引, 在一段时间内易于监测, 单独寻址, 并在需要时检索。液滴微阵列可以实现高密度的微型反应堆, 每个芯片上有数以千计的元素, 它们要么是自由的, 要么由 microwell 结构支撑。18,19,20可通过液体搬运机器人、喷墨检举器、接触 microarrayers21、22、23、24、25等顺序沉积形成 26或者他们可以自组装在表面上, 如 superhydrophillic 斑点在超疏水性表面图案。27,28,29
考虑到这些因素, 可寻址液滴微阵列 (ADMs) 设计为将微滴微阵列的多功能性、空间寻址和体积减小与单分子 TIRF 显微镜的灵敏度相结合, 以定量测量蛋白质丰度。5 ADMs 使单细胞分析形成一个含有单细胞的液滴微阵列在抗体微阵列上, 然后用油覆盖以防止蒸发。水滴的体积是离散的, 以防止样本损失, 否则将通过芯片阀门在连续流微流体实现。30单个细胞中靶蛋白的绝对量非常小;然而, 减少的水滴体积允许相对较高的局部浓度, 以便他们被检测使用三明治抗体检测-抗体是固定在一个明显的区域, 或斑点, 在一个表面上, 捕捉蛋白质, 从而绑定荧光标记的检测抗体存在于液滴容积中。作为一个无标签的方法 (即蛋白质靶不需要直接标记), ADMs 一般适用于分析主要来源的细胞, 如处理的血液, 精细需要吸出物和分离的肿瘤活检, 以及细胞从文化和他们的裂解物。
测量细胞中蛋白质丰度的变化在确定反应的异质性方面很重要, 例如药物, 并有助于提供对细胞功能和通路的洞察, 评估亚人口及其行为, 并确定罕见的事件, 否则将掩盖的散装方法。本协议描述了如何生产和使用可寻址的液滴微阵列来定量地测定人类癌细胞中转录因子 p53 的丰度, 并可用于研究 p53 在化疗药物反应中的作用。靶蛋白由捕获和检测抗体的选择决定, 并可被修改以包含更多或不同的目标。提供说明, 以建立一个简单的设备, 其中包含一个同心喷嘴从一般实验室消耗品, 手动阵列 10 nL 油滴。充分的实验过程被描述, 每滴然后被装载与一个单细胞, 然后裂解和蛋白质的表达由单分子决议决定使用 TIRF 显微镜。

<table><tbody><tr><td>&lt;strong&gt;Cell culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Life Technologies</td><td>10010015</td><td></td></tr><tr><td>DMEM high glucose</td><td>Sigma</td><td>D6429</td><td></td></tr><tr><td>Foetal Bovine Serum (FBS)</td><td>Biochrom</td><td>S0115</td><td></td></tr><tr><td>cell culture flasks</td><td>Corning</td><td>SIAL0639</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Biochrom</td><td>L2153</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microarray&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microcontact Arrayer</td><td>DigiLab, UK</td><td>OmniGrid Micro</td><td></td></tr><tr><td>Microcontact pin</td><td>ArrayIt, USA</td><td>946MP2</td><td></td></tr><tr><td>Coverslips (Nexterion)</td><td>Schott, Europe</td><td>1098523</td><td>Size (mm): 65.0 x 25.0; Thickness (mm) 0.17</td></tr><tr><td>p53 capture antibody</td><td>Enzo</td><td>ADI-960-070</td><td></td></tr><tr><td>p53 detection antibody, Alexa Fluor 488 labelled</td><td>Santa Cruz</td><td>sc-126</td><td>stock concentration 200&amp;mu;g/mL</td></tr><tr><td>Saline-sodium citrate buffer</td><td>Gibco</td><td>15557-044</td><td></td></tr><tr><td>Betaine</td><td>Sigma</td><td>61962</td><td></td></tr><tr><td>Sodium dodecyl sulphate</td><td>Sigma</td><td>L3771</td><td></td></tr><tr><td>384 well plate (low volume)</td><td>Sigma</td><td>CLS4511</td><td></td></tr><tr><td>Nitrogen gas cylinder</td><td>BOC</td><td>Industrial grade, oxygen-free</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Droplets&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micromanipulator</td><td>Eppendorf</td><td>Patchman NP2</td><td></td></tr><tr><td>Manual Microinjector</td><td>Eppendorf</td><td>CellTram Vario</td><td></td></tr><tr><td>Micropipette</td><td>Origio, Denmark</td><td>MBB-FP-L-0</td><td></td></tr><tr><td>Syringe pumps</td><td>KD Scientific</td><td>KDS-210</td><td></td></tr><tr><td>100 &amp;mu;L syringe</td><td>Hamilton</td><td>81020</td><td>Gas tight, PTFE Luer lock</td></tr><tr><td>1 mL syringe</td><td>Hamilton</td><td>81327</td><td>Gas tight, PTFE Luer lock</td></tr><tr><td>Silicone isolator</td><td>Grace Bio-Labs</td><td>JTR24R-A-0.5</td><td>6x4 well silicone isolator with adhesive</td></tr><tr><td>Laser cutter</td><td>VersaLASE</td><td>VLS2.30 CO2 Laser 3W</td><td>for laser cutting of custom isolators</td></tr><tr><td>1mm thick acrylic sheet</td><td>Weatherall-UK</td><td>Clarex Precision Sheet 001</td><td>for laser cutting of custom isolators</td></tr><tr><td>Adhesive sheet</td><td>3M</td><td></td><td>used to adhere custom isolators to microarrayed coverslips</td></tr><tr><td>Super glue</td><td>Loctite</td><td>LOCPFG3T</td><td></td></tr><tr><td>150 &amp;mu;m ID/360 &amp;mu;m OD fused silica tubing</td><td>IDEX</td><td>FS-115</td><td></td></tr><tr><td>1.0 mm ID/1/16&amp;rdquo; OD PFA tubing</td><td>IDEX</td><td>1503</td><td></td></tr><tr><td>0.014&amp;rdquo; ID/0.062&amp;rdquo; OD PTFE tubing</td><td>Kinesis</td><td>008T16-100</td><td></td></tr><tr><td>1.0 mm ID/2.0 mm OD FEP tubing</td><td>IDEX</td><td>1673</td><td></td></tr><tr><td>Bovine Serum Albumen (BSA)</td><td>Fisher Scientific</td><td>BP9700100</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M5904</td><td></td></tr><tr><td>Ultra-pure water</td><td>Millipore, Germany</td><td>MilliQ</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscopy &amp;amp; Optics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TIRF microscope with encoded XY stage</td><td>Nikon, Japan</td><td>Nikon Ti-E</td><td></td></tr><tr><td>EM-CCD</td><td>Andor Technologies, Ireland</td><td>IXON DU-897E</td><td></td></tr><tr><td>Laser excitation source</td><td>Vortran, USA</td><td>Stradus 488-50</td><td></td></tr><tr><td>Optical lysis laser source</td><td>Continuum, USA</td><td>Surelite SLI-10</td><td></td></tr><tr><td>Microscope filter cube for TIRF</td><td>Chroma, USA</td><td>z488bp</td><td></td></tr><tr><td>Microscope filter cube for Optical Lysis</td><td>Laser 2000, UK</td><td>LPD01-532R-25</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fiji</td><td>Open Source</td><td></td><td>Image analysis software</td></tr><tr><td>Matlab</td><td>Mathworks</td><td>version 7.14 or higher</td><td>Image analysis software</td></tr></tbody></table>

counting proteins in single cells with addressable droplet microarrays

通常, 在人口层面分析细胞行为和细胞反应, 在这种情况下, 许多细胞的反应汇集在一起, 平均结果掩盖了复杂人群中丰富的单细胞行为。单细胞蛋白检测和定量技术近年来取得了显著的效果。在这里, 我们描述了一个实用的和灵活的单细胞分析平台, 基于可寻址滴微阵列。本研究描述了如何用单细胞分辨率测量靶蛋白的绝对拷贝数。抑癌 p53 是人类癌症中最常见的突变基因, 超过50% 的癌症病例表现出一种非健康的 p53 表达模式。该协议描述了创建10个 nL 液滴的步骤, 其中单独的人类癌细胞被隔离, p53 蛋白的拷贝数用单分子分辨率测量, 以精确确定表达的变异性。该方法可应用于任何细胞类型, 包括主要材料, 以确定任何感兴趣的目标蛋白的绝对拷贝数。

p53 的绝对基底蛋白拷贝数是在人结肠癌细胞系中以单细胞分辨率确定的, 是细胞。我们展示了 p53 的表达如何在几个数量级上变化, 并显示在静止的细胞群中细胞大小和蛋白质复制数之间的弱正相关。
可寻址液滴微阵列是当水滴在抗体斑点位置, 并以油为上限时形成的。在这里, 水滴支持一个敏感的 p53 蛋白检测。盖玻片使用接?...

Watch this Scientific Journal Video about Counting Proteins in Single Cells with Addressable Droplet Microarrays at JoVE.com

Counting Proteins in Single Cells with Addressable Droplet Microarrays

在这里, 我们提出了可寻址的液滴微阵列 (ADMs), 一种能够确定单细胞中绝对蛋白质丰度的液滴数组方法。我们展示了 ADMs 的能力来表征肿瘤抑制蛋白 p53 在人类癌细胞系中表达的异质性。

用可寻址滴微阵列计算单细胞中的蛋白质

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

counting-proteins-in-single-cells-with-addressable-droplet-microarrays

Research

JoVE Journal

Cancer Research

8.5K 次观看.  Imperial College London. 在这里, 我们提出了可寻址的液滴微阵列 (ADMs), 一种能够确定单细胞中绝对蛋白质丰度的液滴数组方法。我们展示了 ADMs 的能力来表征肿瘤抑制蛋白 p53 在人类癌细胞系中表达的异质性。

视频: Counting Proteins in Single Cells with Addressable Droplet Microarrays

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

科研

生物工程

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

高密度条码抗体芯片的水流模式的指导下制作

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

化学

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

生物化学

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

用于从单一DNA分子大规模平行蛋白质合成的Femtoliter滴体阵列

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

无交叉反应和无免疫多路复用夹芯芯片

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

<meta charset="utf8"></head><body>用于单细胞因子分泌分析的微流控液滴测定

Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. This response involves numerous immune cells secreting various cytokines to orchestrate the immune reaction. However, the secretion dynamics, amounts, and co-occurrence of the different cytokines by various cell subtypes remain poorly understood due to a lack of appropriate tools to study them. Here, we describe a protocol using a microfluidic droplet platform that allows the time-resolved quantitative measurement of secretion dynamics for several cytokines in parallel on the single-cell level. This is enabled by the encapsulation of individual cells into microfluidic droplets together with a multiplexed immunoassay for parallel quantification of cytokine concentrations, their immobilization for dynamic fluorescent imaging, and the analysis of the respective images to derive secreted quantities and dynamics. The protocol describes the preparation of functionalized magnetic nanoparticles, calibration experiments, cell preparation, and the encapsulation of the cells and nanoparticles into droplets for fluorescent imaging and subsequent image and data analysis using the example of lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The presented platform identified distinct cytokine secretion behavior for single and co-secreting cells, characterizing the expected phenotypic heterogeneity in the measured cell sample. Furthermore, the modular nature of the assay allows its adaptation and application to study a variety of proteins, cytokines, and cell samples, potentially leading to a deeper understanding of the interplay between different immune cell types and the role of the different cytokines secreted dynamically to shape the tightly regulated immune response. These new insights could be particularly interesting in the studies of immune dysregulations or in identifying target populations in therapy and drug development.

<meta charset="utf8"></head><body>作者聚焦：揭示免疫反应中的多功能性和异质性

The protocol describes an advanced microfluidic platform to quantitatively measure cytokine secretion dynamics of individual human peripheral blood mononuclear cells. The platform measures up to three cytokines in parallel (IL-6, TNF&#945;, and IL-1&#946;) for each individual cell stimulated with lipopolysaccharide as an example.

微流控方法解决单个多功能细胞的同时和连续细胞因子分泌 

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. ...

用可寻址滴微阵列计算单细胞中的蛋白质

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