Name: counting proteins in single cells with addressable droplet microarrays
Uploaded: 2018-07-06T13:00:00
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
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制作芯片和打印抗体微阵列
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<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?...

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	<li>Willison, K. R., Klug, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+single+cell+and+single+molecule+proteomics+for+clinical+studies.">Quantitative single cell and single molecule proteomics for clinical studies.</a> Curr. Opin. Biotechnol. 24 (4), 745-751 (2013).</li><li>Heath, J. R., Ribas, A., Mischel, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+tools+for+drug+discovery+and+development.">Single-cell analysis tools for drug discovery and development.</a> Nat. Rev. Drug Discov. 15 (3), 204-216 (2016).</li><li>Eyer, K., Stratz, S., Kuhn, P., K&#252;ster, S. K., Dittrich, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementing+enzyme-linked+immunosorbent+assays+on+a+microfluidic+chip+to+quantify+intracellular+molecules+in+single+cells.">Implementing enzyme-linked immunosorbent assays on a microfluidic chip to quantify intracellular molecules in single cells.</a> Anal. Chem. 85 (6), 3280-3287 (2013).</li><li>Salehi-Reyhani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+first+step+towards+practical+single+cell+proteomics:+a+microfluidic+antibody+capture+chip+with+TIRF+detection.">A first step towards practical single cell proteomics: a microfluidic antibody capture chip with TIRF detection.</a> Lab. Chip. 11 (7), 1256-1261 (2011).</li><li>Salehi-Reyhani, A., Burgin, E., Ces, O., Willison, K. R., Klug, D. 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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>Cell culture</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microarray</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&#956;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Droplets</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 &#956;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 &#956;m ID/360 &#956;m OD fused silica tubing</td>
			<td>IDEX</td>
			<td>FS-115</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 mm ID/1/16&#8221; OD PFA tubing</td>
			<td>IDEX</td>
			<td>1503</td>
			<td></td>
		</tr>
		<tr>
			<td>0.014&#8221; ID/0.062&#8221; 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopy &#38; Optics</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</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

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Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

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癌症研究

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Desthiobiotin-链亲和素亲和介导纯化间皮瘤细胞 RNA 相互作用蛋白的研究

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

磷流式细胞仪与荧光细胞条形码用于单细胞信号分析和生物标志物发现

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

利用微阵列研究微环境对癌症细胞表型的影响

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

矫形子宫内膜癌模型，具有从 Vivo 传播和培养的 VX2 细胞中制成的再生淋巴病

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

液滴数字 TRAP (ddTRAP): 对端粒重复放大协议的适应性, 以实现液滴数字聚合酶链反应

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

斑马鱼中具有功能化荧光硅纳米粒子的异种移植人类癌细胞的Vivo靶向

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

使用宽场活细胞成像进行批量和单类器官分析的多参数肿瘤类器官药物筛选

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Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

小鼠患者来源的癌细胞原位植入概括了晚期结直肠癌

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