通过玻璃表面功能靶细胞提取方法
Summary
This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.
Abstract
One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.
Introduction
当前台式细胞分离方法（ 如荧光活化细胞分选1，激光捕获显微解剖2，免疫磁珠分离1）可能需要几个小时的准备和排序的。这些大的时间尺度可影响生理反应和表达水平，导致在不能代表生理反应3的分析。系统需要能够迅速和有效地分离出特定的细胞类型，而为了提高生物医学应用细胞分离和富集破坏细胞表面受体的水平。因此，我们的做法的理由是开发细胞分离温和的做法。
该“芯片实验室”的理念，提供更快的幅度（小时到分钟）细胞分离的订单的承诺，最经常涉及捕捉到细胞表面和释放细胞或细胞内的小故事通过物理4,5 NTS或化学方法6。虽然这些方法提供了一些优点，如鉴定蛋白质7,8-表达，识别RNA表达9-11，或甚至提供用于在体外培养12,13细胞，许多这些技术不能被转换为诊断如细胞受体分析因到它们的非生理环境。酶促起重剂如胶原酶也可影响这些受体数量14,15，这意味着使用这些起重剂不会产生准确的生理数据细胞受体定 量技术。细胞裂解防止分化的原始表面受体之间，以及那些以前内在16。本协议描述了细胞分离快速，轻柔的手法。
Protocol
1.清洁玻璃表面和试剂的准备放置一个玻璃表面在氧等离子体机，在50％的功率进行清洁5分钟。 制作2.5毫升2-％重构（3-氨基丙基）三乙氧基硅烷（APTES）溶液中，加入APTES 50微升并在锥形管2.45毫升乙醇中。 2. APTES和DSB功能化添加APTES溶液到表面。每孔吸取150微升8孔板。每孔移液器将100μl24孔板中。吸取1.1毫升60×15毫米的玻璃菜肴。覆盖表面，防止蒸…
Representative Results
使用该协议，我们表明细胞捕获（ 图3A）和MCF7GFP细胞的细胞释放（ 图3C）以及活细胞对照（ 图4）。我们量化的细胞捕获为60％和80％被释放（ 图3C）。当我们扩展这种方法的RAW 264.7巨噬细胞和MCF7GFP细胞的混合物，RAW巨噬细胞的50％被捕获（ 图3D）和RAW巨噬细胞有80％是释放用20mM生物素（ 图3B）。…
Discussion
在细胞分离技术的进步，进一步加强结构与功能的关系科学的研究在神经科学18，遏制再生生物学血管生物学19单元编程和血管生成的信号。事实上，原代细胞培养20（ 例如 ，内皮细胞）在血管生物学主要通过使用细胞分离技术进行。细胞分离最近还被用于细胞膜受体3,14,15,19,21定量流量（qFlow）仪分析。但是，现有的细胞分离方法，影响细胞表面受体的水平…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We would like to thank the American Cancer Society, Illinois Division (282802) and the National Science Foundation CBET (1512598) for funding support. We also would like to thank Dr. Dianwen Zhang from the University of Illinois Beckman Institute for microscopy training. Finally, we would like to thank Jared Weddell, Stacie Chen, and Spencer Mamer for insightful discussions.
Materials
| (3-Aminopropyl) triethoxysilane (APTES) | Acros Organics | 919-30-2 | Used to make 2% APTES solution |
| Plasma Cleaner Pico | Diener | Model 1 | Cleans surfaces and allows for bonding of PDMS to glass |
| d-Desthiobiotin (DSB) | Sigma | D20655 | Used as the releasing mechanism in the cellular capture surface. |
| dimethyl sulfoxide (DMSO) | British Drug Houses (BDH) | BDH1115-1LP | Dissolves the DSB into solution |
| 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) | Thermo-Scientific | 5g: 22980 25g: 22981 |
Activates Carboxylic Acids and allows binding of proteins to glass surface. |
| uncoated 8-well culture slide | BD Falcon | Case of 24: 354118 Case of 96: 354108 |
Used in cellular experiments involving Zeiss fluorescence microscope such as initial capture and release quantification experiments |
| Glass bottom 24-well plates | MatTek | P24G-0-13-F | Used in cellular experiments involving the plate reader such as antibody and cellular titration experiments |
| Mercaptoethanol | Science Lab | 60-24-2 | Used to quench reaction between EDC and DSB |
| 4-Morpholinoethanesulfonic acid hydrate (MES Hydrate 99%) |
Fisher Scientific | AC172590250 | Used to make 0.1 M MES Buffer for use in EDC reaction |
| Precision Oven | Thermo Scientific | 11-475-153 | Used in curing of PDMS and APTES layer. |
| Titramax 1000 Shaker | Heidolph | 13-889-420 | Used to ensure even distribution of APTES on surfaces. |
| 1X Streptavidin 5mg [e7105-5mg] |
Proteo Chem | 9013-20-1 | Biotin-binding protein May cause irritation |
| 5 cm Glass Dish | Fisher Scientific | 08748A | Used in HUVEC studies as well as future profiling studies. |
| 14 cm Petri Dish with Cover | Sigma-Aldrich | Z717231 | Used to hold samples being functionalized and transport them. |
| MCF7-GFP cells | Cell Biolabs | AKR211 | Stored in liquid nitrogen |
| RAW264.7 mouse macrophages |
ATCC | TIB-71 | Gifted to us from Smith lab at the University of Illinois. Stored in liquid nitrogen |
| TrypLE | Life Technologies | 12605036 | Stored in 100mL at room temperature |
| Dulbecco’s modified Eagle medium | Cell Media Facility at School of Chemical Sciences at UIUC | 50003PC | Supplier: Corning |
| Nonessential amino acids | Cell Media Facility at School of Chemical Sciences at UIUC | 25-025-CI | Already added into DMEM by facility. Supplier: Corning |
| 10% fetal bovine serum | Fisher Scientific | 03-600-511 | Stored in 500mL at < -10⁰C |
| 1% Penicillin–Streptomycin | Life Sciences Storeroom at UIUC | 17602e | Supplier: VWR Stored in 100 ml at 4⁰C |
| Cell scraper | Fisher Scientific | 12-565-58 | Small 23cm 50 pack |
| Cell Dissociation Solution | Corning | MT-25-056CI | Used to lift cells non-enzymatically for the use in cell experiments |
| Hemacytometer | Hausser | 02-671-54 | Used to count cells for quantification of cell solutions and capture and release effectivity. |
| Biotin | Amresco | 58-85-5 | Used to release cells from surface. |
| HBSS | Created from Recipe | N/A | Used to keep cells alive in suspension as well as wash surfaces of non-specific binding. (Adapted from Cold Spring Harbor Protocols): In 500 mL, use 4 g NaCl, .2 g KCl, .0402 g Na2PO4*7H2O, .03 g KH2PO4 and .5 g Glucose. Add DI water to get to 500 mL, filter, and then refrigerate. |
| HLA-ABC Antibody | BioLegend | 311402 | Antibody used to capture MCF7gfp cells |
| hIgG Antibody | BioLegend | HP6017 | Antibody used to capture MCF7gfp cells |
| MCF7 GFP cells | Cell Biolabs | AKR-211 | Luminal Breast Cancer line that has been transfected with green fluorescent protein. |
| Assorted Conicals | Thermo-Scientific | 15mL: 12-565-268 | 50/15 mL plastic conicals for storing solutions and aliquots. |
| Mini-Tube Rotators (End over End Mixer) | Fisher Scientific | 05-450-127 | Used to incubate antibody and mix other cellular solutions in order to mix |
| Axiovert 200M (Fluorescence Microscope) | Zeiss | N/A | Zeiss Axiovert 200 M inverted florescence microscope. |
| Zeba Desalting columns | Thermo-Scientific | PI-87770 | Used to purify newly biotinylated antibodies after the use of the Biotinylation Kit. Instructions provided at: http://www.funakoshi.co.jp/data/datasheet/PCC/89894.pdf |
| EZ Link Sulfo NHS Low Weight Biotinylation Kit | Thermo- Scientific | Used to biotinylate antibodies to allow them to integrate with the capture surface | |
| Plate Reader | BioTek | Synergy HTX Multimode Reader | Used to quantitatively measure fluorescent intensity in the titration experiments. |
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Citazione di questo articolo
Ansari, A., Patel, R., Schultheis, K., Naumovski, V., Imoukhuede, P. I. A Method of Targeted Cell Isolation via Glass Surface Functionalization. J. Vis. Exp. (115), e54315, doi:10.3791/54315 (2016).