使用磁悬浮定量细胞密度和抗原特性
Summary
本文描述了一种基于磁悬浮的方法，该方法可以通过量化具有固定密度的捕获微球悬浮高度的变化来特异性检测抗原的存在，无论是可溶的还是膜结合的。
Abstract
所描述的方法是基于磁悬浮原理开发的，磁悬浮原理根据其密度和磁性分离细胞和颗粒。密度是一种识别特性的细胞类型，与其代谢率、分化和活化状态直接相关。磁悬浮允许一步法成功分离，成像和表征循环血细胞，并根据密度和磁性检测贫血，镰状细胞病和循环肿瘤细胞。这种方法也适用于通过使用分别包被捕获和检测抗体的低密度和高密度微球组来检测溶液中存在的可溶性抗原。如果抗原存在于溶液中，它将桥接两组磁珠，产生新的磁珠复合物，该复合物将悬浮在抗体包被的磁珠行之间。与较低浓度的抗原相比，增加溶液中靶抗原的浓度将产生更多的珠状 – 珠状复合物，从而允许对靶抗原进行定量测量。磁悬浮比其他方法有利，因为它减少了样品制备时间并且缺乏对经典读出方法的依赖性。生成的图像可使用标准显微镜或移动设备（如智能手机或平板电脑）轻松捕获和分析。
Introduction
磁悬浮是一种技术，仅用于分离，分析和鉴定细胞类型1，2，3，蛋白质4，5和阿片类药物6，仅基于其特异性密度和顺磁性。细胞密度是每种细胞类型独特的内在属性，直接关系到其代谢率和分化状态7、8、9、10、11、12、13、14。量化稳态条件下和各种细胞过程中细胞密度的细微和瞬态变化，可以为人们提供对细胞生理学和病理生理学无与伦比的洞察力。细胞密度的变化与细胞分化15、16、细胞周期进展9、17、18、19、凋亡20、21、22、23和恶性转化24、25、26有关。.因此，量化细胞密度的特定变化，可用于区分不同类型的细胞，以及区分经历各种活化过程的相同类型的细胞。这使得针对特定细胞亚群的实验成为可能，其中密度的动态变化作为细胞代谢改变的指标27。由于已经确定细胞可以响应于不断变化的环境7而改变其密度，因此必须测量细胞的动力学与其密度的关系以充分理解它，而目前的方法可能无法提供12。另一方面，磁悬浮允许对细胞及其性质进行动态评估28。
电池是抗磁性的，这意味着它们没有永久的磁性偶极矩。然而，当暴露于外部磁场时，在电池中产生弱磁偶极矩，与外加磁场的方向相反。因此，如果细胞悬浮在顺磁性溶液中并暴露在强烈的垂直磁场中，它们将悬浮在远离磁源的位置并停止到一定高度，这主要取决于它们的个体密度。当满足以下两个标准时，可以将物体的抗磁性悬浮限制在不均匀磁场的最小值：1）粒子的磁化率必须小于周围介质的磁化率，以及2）磁力必须足够强以抵消粒子的浮力。通过将红细胞悬浮在磁性缓冲液中，并使用小的、廉价的、市售的永磁体产生强磁场梯度，可以满足这两个标准1。磁性捕获粒子在沿重力方向的轴上的平衡位置由其密度（相对于缓冲区的密度）、磁化率（相对于缓冲区的磁化率）和所施加磁场的特征决定。由于溶液的密度和磁性在整个系统中是恒定的，因此细胞的固有密度特性将是决定细胞悬浮高度的主要因素，与密度较低的细胞相比，密度较大的细胞悬浮得更低。这种方法使用一组两个密度参考珠（1.05和1.2 g / mL），使我们能够使用精确的比例分析进行密度测量。改变磁性溶液的浓度允许人们分离不同的细胞群，例如红细胞和WBC，因为循环细胞的密度是细胞特异性的，从而无需分离方案或其他细胞操作。
生物学研究中使用的大多数检测方法依赖于将特定结合事件外推为易于量化的线性信号。这些读出方法通常很复杂，涉及专门的设备和专门的科学人员。本文描述了一种旨在检测在细胞或细胞外囊泡的质膜上发现的抗原或可溶于血浆的抗原的方法，使用一种或两种抗体包被的微球。这些珠子彼此之间以及被审问目标的密度必须不同。在任何给定的生物流体中靶抗原的存在被翻译成与检测珠结合的抗原阳性细胞悬浮高度的特定，可测量的变化。在可溶性抗原或细胞外囊泡的情况下，它们与捕获和检测微球结合，形成珠状 – 珠复合物而不是珠细胞复合物。悬浮高度的变化取决于磁珠细胞或磁珠-珠复合物的新密度。除了配合物悬浮高度的变化，这表明生物流体中存在抗原外，复合物的数量还取决于靶标的量，使得磁悬浮也成为抗原检测的定量方法24。
Protocol
本研究中使用的实验方案已获得Beth Israel Deaconess医疗中心机构审查委员会（IRB）的批准。 1. 仪器设置 注意：成像悬浮电池需要两个在z轴上磁化的稀土钕磁铁放置在彼此相对的同一极上以产生磁场。磁铁之间的距离可以根据磁场的强度和目标的密度进行定制。在这种情况下，磁体由1mm的空间隔开，足以插入50 mm长的1×1 mm方形玻璃毛细管。该设备使用AutoCAD设?…
Representative Results
磁悬浮根据物体的密度、其磁性特征、顺磁性溶液的浓度以及两个强大的稀土磁铁产生的磁场强度，将不同密度的物体聚焦在不同的悬浮高度。由于两块磁铁彼此叠加，因此只能使用侧翻的显微镜在保持科勒照明的同时观察悬浮样品（图1）。每种细胞类型达到的最终悬浮高度可以通过改变顺磁性溶液的浓度来轻松修改。图2说明了通过使用不同浓度的?…
Discussion
梯度离心是目前根据其独特密度分离亚细胞组分的标准技术。然而，这种方法需要使用专门的梯度介质以及离心机设备。这里介绍的磁悬浮方法允许详细研究循环细胞的形态学和功能特性，对细胞进行最小的操作（如果有的话），提供对循环细胞 的近体内 访问。
但是，在使用磁悬浮时，有几个点值得一提。首先，用于成像的显微镜必须专用于这种方法，因为设置非常…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者要感谢Getulio Pereira博士对细胞外囊泡工作的帮助。
这项工作得到了以下国家卫生研究院对ICG的资助：RO1CA218500，UG3HL147353和UG3TR002881。
Materials
| 2-(N-Morpholino)ethanesulfonic acid hydrate | Sigma Aldrich | M-2933 | (MES); component of activation buffer |
| 50×2.5×1 mm magnets, Nickel (Ni-Cu-Ni) plated, grade N52, magnetized through 5mm (0.197") thickness | K&J Magnetics | Custom | Magnets used for the magnetic levitation device |
| Capillary Tube Sealant (Critoseal) | Leica Microsystems | 267620 | Used to cap the ends of the capillary tubes |
| Centrifuge tube filters (Corning Costar Spin-X) | Sigma Aldrich | CLS8163 | Used to wash beads |
| Compact Lab Jack | Thorlabs | LJ750 | Used for adjusting the magnetic levitation device |
| DPBS, no calcium, no magnesium | Gibco | 14190-144 | Solution for bead suspensions |
| Ethanolamine | Sigma Aldrich | E9508-100ML | Used during a wash step for beads |
| Fluorescent Plasma Membrane Stain (CellMask Green) | Invitrogen | C37608 | Used to stain Rh+ cells |
| Gadoteridol Injection | ProHance | NDC 0270-1111-03 | Gadolinium (Gd3+); magnetic solution used to suspend cells |
| HBSS++ | Gibco | 14025-092 | Solution for sample preparation |
| Human C5b,6 complex | Complement Technology, Inc | A122 | Used to generate RBC Evs |
| Human C7 protein | Complement Technology, Inc | A124 | Used to generate RBC Evs |
| Human C8 protein | Complement Technology, Inc | A125 | Used to generate RBC Evs |
| Human C9 protein | Complement Technology, Inc | A126 | Used to generate RBC Evs |
| Mini Series Post Collar | Thorlabs | MSR2 | Used to secure magnetic levitation device to lab jacks |
| N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride | Sigma Aldrich | E1769-10G | (EDC); used in antibody coupling reaction |
| Normal Rabbit IgG Control | R&D Systems | AB-105-C | Used to coat beads as a control condition |
| Phosphate Buffered Saline (10X Solution, pH 7.4) | Boston Bioproducts | BM-220 | Component of coupling buffer, used for washing steps |
| Polysorbate 20 (Tween 20) | Sigma Aldrich | P7949-500ML | Component of activation buffer |
| Polystyrene Carboxyl Polymer | Bangs Laboratories | PC06004 | Top density beads (1.05 g/mL), used for antibody coupling |
| Rabbit RhD Polyclonal Antibody | Invitrogen | PA5-112694 | Used to coat beads for the dectection of Rh factor in red blood cells |
| Research Grade Microscope | Olympus | Provis AX-70 | Microscoped used to mount magnetic levitation device and view levitating cells |
| Rubber Dampening Feet | Thorlabs | RDF1 | Used to support the breadboard table |
| Square Boro Tubing | VitroTubes | 8100-050 | Capillary tube used for loading sample into Maglev |
| Sulfo-NHS | Thermoscientific | 24510 | Used in antibody coupling reaction |
| Translational Stage | Thorlabs | PT1 | Used for focusing and for scanning capillary tube |
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Citar este artigo
Thompson, L., Pinckney, B., Lu, S., Gregory, M., Tigges, J., Ghiran, I. Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation. J. Vis. Exp. (171), e62550, doi:10.3791/62550 (2021).