原子力显微镜罐体功能化,具有单T细胞或单粒子免疫单细胞力光谱
Summary
我们提出了一种协议,用单个T细胞和珠子粒子使原子力显微镜(AFM)的吊带功能化,用于免疫学研究。显示了通过AFM探测单对T细胞树突状细胞结合的过程,并监测宏噬细胞对单个固体颗粒的实时细胞响应,并采用荧光成像。
Abstract
基于原子力显微镜的单细胞力光谱(AFM-SCFS)是研究活细胞生物物理特性的有力工具。这种技术允许在活细胞膜上探测相互作用强度和动力学,包括细胞、受体和配体之间的相互作用强度和动力学,以及许多其他变异。它还作为一种机制,以时空控制的方式在单个细胞上提供物理或生化刺激,从而在与活细胞结合时实时监控特定的细胞激活和随后的细胞事件荧光成像。这些 AFM-SCFS 测量的关键步骤是 AFM 悬臂功能化,换句话说,将感兴趣的主题附加到悬臂上。在这里,我们提出用单个T细胞和单个聚苯乙烯珠分别修改AFM吊杆的方法,用于免疫学研究。前者涉及一种生物相容性胶水,将单T细胞耦合到溶液中的扁平悬臂尖端,而后者则依靠环氧胶在空气环境中的单珠粘附。还提供了与每个悬臂修饰相关的两个免疫学应用。此处描述的方法可以很容易地适应不同的细胞类型和固体颗粒。
Introduction
原子力显微镜(AFM),一个多功能的工具,在细胞生物学研究1,2,3,4,5中已经发现许多应用。除了高分辨率成像能力外,原生力探测功能还允许活细胞的生物物理特性在单细胞6、7级直接就地研究。这些包括亚细胞结构,甚至整个细胞的刚性 8,9,10,11,12,特定的配体/受体结合强度细胞表面的单分子水平13,固体粒子和细胞的单对之间的附着力或两个细胞1、2、14、15之间的附着力。后两种通常被归类为单细胞力光谱(SCFS)16。由于各种弹簧常数的易得的悬臂,AFM 可访问的力范围相当广泛,从几皮克纽顿 (pN) 到微牛顿 (μN),这足以覆盖涉及几十个力的整个细胞事件范围pN,如基于受体的单分子结合,到nN,如噬菌体细胞事件15。这种较大的动态力范围使 AFM 优于其他力探测技术,如光学/磁性钳子和生物膜力探头,因为它们更适合弱力测量,其力通常小于 200 pN17,18.此外,AFM可以作为高精度操纵器,以临时定义的方式4,19向单个细胞提供各种刺激。这是理想的实时单细胞活化研究。结合活细胞荧光成像,可以同时监测随后细胞对特定刺激的反应,从而使基于AFM的SCFS作为光学成像极其强大,为探测细胞信号提供了实用的工具。例如,AFM用于确定在成骨菌20中引起钙瞬变所需的菌株。在这项工作中,在用AFM尖端在培养的成骨器上应用局部力后,通过钙比例成像对钙瞬变进行荧光跟踪。最近,AFM被用于拉伸胶原纤维,在胶原纤维上生长肝硬质细胞(HSC),这种经研究用的HSC活化由荧光Src生物传感器实时监测,其磷酸化代表生物传感器的荧光强度与HSC活化3相关。
在基于 AFM 的 SCFS 实验中,AFM 悬臂的正确功能化是成功测量的关键步骤。由于我们的研究兴趣集中在免疫细胞激活,我们经常功能与颗粒物质,如单个固体颗粒,可以触发噬菌体和/或强烈的免疫反应4,14,15和单个T细胞,可以形成免疫突触与抗原呈现细胞,如活化树突细胞(DC)2。单固体颗粒通常通过空气环境中的环氧胶与悬臂耦合,而单 T 细胞由于其非粘合性质,通过溶液中的生物相容性胶水功能化为悬臂。在这里,我们将描述执行这两种类型的悬臂修改的方法,并给出两个关联的应用程序。第一个应用是探测T细胞/直流与AFM-SCFS的相互作用,以便从细胞力学的角度理解调节T细胞的抑制机制。第二个涉及将AFM与活细胞荧光成像相结合,实时监测巨噬细胞对固体颗粒的细胞反应,揭示受体独立磷脂醇4,5-双磷酸(PIP2)的分子机制-莫辛调解了方位体。该协议的目的是为感兴趣的研究人员提供一个参考框架,以设计和实现自己的实验设置与基于AFM的单细胞分析的免疫学研究。
Protocol
小鼠实验方案遵循清华大学动物护理指南 1. 单T细胞的悬臂功能化 小鼠脾细胞制剂 牺牲小鼠(8-16周的年龄(两性);例如,C57BL/6菌株)使用二氧化碳,其次是宫颈脱位。 用75%乙醇清洁小鼠,进行中线皮肤切口,然后进行切除。 使用玻璃滑梯将含有 2% 胎儿牛血清 (FBS) 的 PBS 4 mL 中的脾脏均匀化,并通过 70 μm 网状尼龙滤网将细胞悬浮液排出骨质和碎屑。 <…
Representative Results
图 4A显示了单 T 单元和单 DC 之间在一个接近缩回周期中结合交互的典型力距离曲线。浅红色曲线是延伸曲线,深红色曲线为回缩曲线。由于延伸曲线通常用于缩进或刚度分析,因此这里只涉及缩进曲线, 与细胞粘附有关。曲线中的最小值(绿色圆圈)表示最大附着力的度量。曲线下的区域(带沙区)表示将 T 单元与 DC 分离所需的工作(能量)。在完全分离之前,…
Discussion
基于AFM的单细胞力光谱已经演变为解决活细胞生物物理特性的有力工具。对于这些应用,悬臂需要正确工作,以便探测感兴趣的单元上的特定交互或属性。分别介绍了单T单元和单微米尺寸珠与无尖悬臂耦合的方法。为了将单个T细胞附着在悬臂上,选择生物相容性胶水作为细胞粘合剂。它是一种从海洋贝类中提取的特制蛋白质溶液。其粘附性源于多酚残留物,其羟基基组可与以非特异性方式在细胞表面?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金总体计划(31370878)、国家重点计划(31630023)和创新研究组计划(81621002)的支持。
Materials
| Material | |||
| 10 μl pipette tip | Thermo Fisher | 104-Q | |
| 15 ml tube | Corning | 430791 | |
| 6 cm diameter culture dish | NALGENE nunc | 150462 | |
| 6-well culture plate | JET | TCP011006 | |
| AFM Cantilever | NanoWorld | Arrow-TL1-50 | tipless cantilever |
| β-Mercaptoethanol | Sigma | 7604 | |
| Biocompatible glue | BD Cell-Tak | 354240 | |
| CD4+ T cell isolation Cocktail | STEMCELL | 19852C.1 | |
| DC2.4 cell line | A gift from K. Rock (University of Massachusetts Medical School, Worcester, MA) | ||
| Dextran-coated magnetic particles | STEMCELL | SV30010 | |
| EDTA | GENEray | Generay-E1101-500 ml | |
| Epoxy | ERGO | 7100 | |
| Ethanol | twbio | 00019 | |
| FBS | Ex Cell Bio | FSP500 | |
| FcR blocker | STEMCELL | 18731 | |
| Glass coverslip | local vender (Hai Men Lian Sheng) | HX-E37 | 24mm diameter, 0.17mm thinckness |
| Glass slides | JinTong department of laboratory and equipment management, Haimen | N/A | customized |
| H2O2 (30%) | Sino pharm | 10011218 | |
| H2SO4 | Sino pharm | 80120892 | |
| HEPES | Sigma | 51558 | |
| Magnet | STEMCELL | 18000 | |
| Mesh nylon strainer | BD Falcon | REF 352350 | |
| Moesin-EGFP | N/A | cloned in laboratory | |
| Mouse CD25 Treg cell positive isolation kit | STEMCELL | 18782 | Component: FcR Blocker,Regulatory T cell Positive Selection Cocktail, PE Selection Cocktail, Dextran RapidSpheres, |
| Mouse CD4+ Tcell isolation kit | STEMCELL | 19852 | Component:CD4+T cell isolation Cocktail, Streptavidin RapidSpheres, Rat Serum |
| NaOH | Lanyi chemical products co., LTD， Beijing | 1310-73-2 | |
| PBS | Solarbio | P1022-500 | |
| PE selection cocktail | STEMCELL | 18151 | |
| Penicillin-Streptomycin | Hyclone | SV30010 | |
| PLCδ-PH-mCherry | Addgene | 36075 | |
| Polystyrene microspheres 6.0μm | Polysciences | 07312-5 | |
| polystyrene round bottom tube | BD Falcon | 352054 | |
| Rat serum | STEMCELL | 13551 | |
| RAW264.7 | ATCC | ||
| Recombinant Human Interleukin-2 | Peprotech | Peprotech, 200-02-1000 | |
| Red blood cell lysis buffer | Beyotime | C3702 | |
| Regulatory T cell positive selection cocktail | STEMCELL | 18782C | |
| RPMI 1640 | Life | C11875500BT | |
| Sample chamber | Home made | ||
| Streptavidin-coated magnetic particles | STEMCELL | 50001 | |
| Transfection kit | Clontech | 631318 | |
| Trypsin 0.25% EDTA | Life | 25200114 | |
| Tweezers | JD | N/A | |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| 20x objective NA 0.8 | Zeiss | 420650-9901 | Plan-Apochromat |
| Atomic force microscope | JPK | cellHesion200 | |
| Centrifuge | Beckman coulter | Allegra X-12R | |
| Fluorescence imaging | home-made objective-type total internal reflection fluorescence microscop based on a Zeiss microscope stand | ||
| Humidified CO2 incubator | Thermo Fisher | HERACELL 150i | |
| Inverted light microscope | Zeiss | Observer A1 manual |
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Cite This Article
Chen, J., Xu, Y., Shi, Y., Xia, T. Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy. J. Vis. Exp. (149), e59609, doi:10.3791/59609 (2019).