Name: quantification of cellular densities and antigenic properties using magnetic levitation
Uploaded: 2021-05-17T15:00:00
Description: Watch this Scientific Journal Video about Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation at JoVE.com

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。

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1x1 mm方形玻璃毛细管。该设备使用AutoCAD设?...

<ol>
	<li>Tasoglu, S., 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=Levitational+Image+Cytometry+with+Temporal+Resolution.">Levitational Image Cytometry with Temporal Resolution.</a> Advanced Materials. 27 (26), 3901-3908 (2015).</li><li>Durmus, N. G., 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=Magnetic+levitation+of+single+cells.">Magnetic levitation of single cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (28), 3661-3668 (2015).</li><li>Knowlton, S., Yu, C. H., Jain, N., Ghiran, I. C., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smart-Phone+Based+Magnetic+Levitation+for+Measuring+Densities.">Smart-Phone Based Magnetic Levitation for Measuring Densities.</a> PLoS One. 10 (8), 0134400 (2015).</li><li>Ashkarran, A. A., Suslick, K. S., Mahmoudi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetically+Levitated+Plasma+Proteins.">Magnetically Levitated Plasma Proteins.</a> Analytical Chemistry. 92 (2), 1663-1668 (2020).</li><li>Yaman, S., Tekin, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+Susceptibility-Based+Protein+Detection+Using+Magnetic+Levitation.">Magnetic Susceptibility-Based Protein Detection Using Magnetic Levitation.</a> Analytical Chemistry. 92 (18), 12556-12563 (2020).</li><li>Abrahamsson, C. K., 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=Analysis+of+Powders+Containing+Illicit+Drugs+Using+Magnetic+Levitation.">Analysis of Powders Containing Illicit Drugs Using Magnetic Levitation.</a> Angewandte Chemie Internation Edition in English. 59 (2), 874-881 (2020).</li><li>Trajkovic, K., Valdez, C., Ysselstein, D., Krainc, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+in+cell+density+alter+protein+markers+of+multiple+cellular+compartments,+confounding+experimental+outcomes.">Fluctuations in cell density alter protein markers of multiple cellular compartments, confounding experimental outcomes.</a> PLoS One. 14 (2), 02117227 (2019).</li><li>Hart, A., Edwards, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buoyant+density+fluctuations+during+the+cell+cycle+of+Bacillus+subtilis.">Buoyant density fluctuations during the cell cycle of Bacillus subtilis.</a> Archives of Microbiology. 147 (1), 68-72 (1987).</li><li>Poole, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+in+buoyant+density+during+the+cell+cycle+of+Escherichia+coli+K12:+significance+for+the+preparation+of+synchronous+cultures+by+age+selection.">Fluctuations in buoyant density during the cell cycle of Escherichia coli K12: significance for the preparation of synchronous cultures by age selection.</a> The Journal General Microbiology. 98 (1), 177-186 (1977).</li><li>Cass, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density-dependent+fluctuations+in+membrane+permeability+in+logarithmically+growing+cell+cultures.">Density-dependent fluctuations in membrane permeability in logarithmically growing cell cultures.</a> Experimental Cell Research. 73 (1), 140-144 (1972).</li><li>Grover, W. H., 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=Measuring+single-cell+density.">Measuring single-cell density.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (27), 10992-10996 (2011).</li><li>Bryan, A. K., 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=Measuring+single+cell+mass,+volume,+and+density+with+dual+suspended+microchannel+resonators.">Measuring single cell mass, volume, and density with dual suspended microchannel resonators.</a> Lab on a Chip. 14 (3), 569-576 (2014).</li><li>Gomer, R. H., Jang, W., Brazill, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+density+sensing+and+size+determination.">Cell density sensing and size determination.</a> Development, Growth and Differentiation. 53 (4), 482-494 (2011).</li><li>Neurohr, G. E., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relevance+and+Regulation+of+Cell+Density.">Relevance and Regulation of Cell Density.</a> Trends in Cell Biology. 30 (3), 213-225 (2020).</li><li>Maric, D., 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=Anatomical+gradients+in+proliferation+and+differentiation+of+embryonic+rat+CNS+accessed+by+buoyant+density+fractionation:+alpha+3,+beta+3+and+gamma+2+GABAA+receptor+subunit+co-expression+by+post-mitotic+neocortical+neurons+correlates+directly+with+cell+buoyancy.">Anatomical gradients in proliferation and differentiation of embryonic rat CNS accessed by buoyant density fractionation: alpha 3, beta 3 and gamma 2 GABAA receptor subunit co-expression by post-mitotic neocortical neurons correlates directly with cell buoyancy.</a> European Journal of Neuroscience. 9 (3), 507-522 (1997).</li><li>Maric, D., Maric, I., Barker, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buoyant+density+gradient+fractionation+and+flow+cytometric+analysis+of+embryonic+rat+cortical+neurons+and+progenitor+cells.">Buoyant density gradient fractionation and flow cytometric analysis of embryonic rat cortical neurons and progenitor cells.</a> Methods. 16 (3), 247-259 (1998).</li><li>Wolff, D. A., Pertoft, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+HeLa+cells+by+colloidal+silica+density+gradient+centrifugation.+I.+Separation+and+partial+synchrony+of+mitotic+cells.">Separation of HeLa cells by colloidal silica density gradient centrifugation. I. Separation and partial synchrony of mitotic cells.</a> Journal of Cell Biology. 55 (3), 579-585 (1972).</li><li>Bienz, K., Egger, D., Wolff, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+replication,+cytopathology,+and+lysosomal+enzyme+response+of+mitotic+and+interphase+Hep-2+cells+infected+with+poliovirus.">Virus replication, cytopathology, and lysosomal enzyme response of mitotic and interphase Hep-2 cells infected with poliovirus.</a> Journal of Virology. 11 (4), 565-574 (1973).</li><li>Baldwin, W. W., Kubitschek, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buoyant+density+variation+during+the+cell+cycle+of+Saccharomyces+cerevisiae.">Buoyant density variation during the cell cycle of Saccharomyces cerevisiae.</a> Journal of Bacteriology. 158 (2), 701-704 (1984).</li><li>Yurinskaya, V. E., 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=Thymocyte+K+,+Na++and+water+balance+during+dexamethasone-+and+etoposide-induced+apoptosis.">Thymocyte K+, Na+ and water balance during dexamethasone- and etoposide-induced apoptosis.</a> Cellular Physiology and Biochemistry. 16 (1-3), 15-22 (2005).</li><li>Wyllie, A. H., Morris, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone-induced+cell+death.+Purification+ad+properties+of+thymocytes+undergoing+apoptosis+after+glucocorticoid+treatment.">Hormone-induced cell death. Purification ad properties of thymocytes undergoing apoptosis after glucocorticoid treatment.</a> American Journal of Pathology. 109 (1), 78-87 (1982).</li><li>Morris, R. G., Hargreaves, A. D., Duvall, E., Wyllie, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone-induced+cell+death.+2.+Surface+changes+in+thymocytes+undergoing+apoptosis.">Hormone-induced cell death. 2. Surface changes in thymocytes undergoing apoptosis.</a> American Journal of Pathology. 115 (3), 426-436 (1984).</li><li>Benson, R. S., Heer, S., Dive, C., Watson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cell+volume+loss+in+CEM-C7A+cells+during+dexamethasone-induced+apoptosis.">Characterization of cell volume loss in CEM-C7A cells during dexamethasone-induced apoptosis.</a> American Journal of Physiology. 270 (4), 1190-1203 (1996).</li><li>Zipursky, A., Bow, E., Seshadri, R. S., Brown, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+density+and+volume+in+normal+subjects+and+in+patients+with+acute+lymphoblastic+leukemia.">Leukocyte density and volume in normal subjects and in patients with acute lymphoblastic leukemia.</a> Blood. 48 (3), 361-371 (1976).</li><li>Huber, C., 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+comparative+study+of+the+buoyant+density+distribution+of+normal+and+malignant+lymphocytes.">A comparative study of the buoyant density distribution of normal and malignant lymphocytes.</a> British Journal of Haematology. 40 (1), 93-103 (1978).</li><li>Griwatz, C., Brandt, B., Assmann, G., Zanker, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunological+enrichment+method+for+epithelial+cells+from+peripheral+blood.">An immunological enrichment method for epithelial cells from peripheral blood.</a> Journal of Immunological Methods. 183 (2), 251-265 (1995).</li><li>Andersen, M. S., 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+Novel+Implementation+of+Magnetic+Levitation+to+Quantify+Leukocyte+Size,+Morphology,+and+Magnetic+Properties+to+Identify+Patients+with+Sepsis.">A Novel Implementation of Magnetic Levitation to Quantify Leukocyte Size, Morphology, and Magnetic Properties to Identify Patients with Sepsis.</a> Shock. , (2018).</li><li>Felton, E. J., 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=Detection+and+quantification+of+subtle+changes+in+red+blood+cell+density+using+a+cell+phone.">Detection and quantification of subtle changes in red blood cell density using a cell phone.</a> Lab on a Chip. 16 (17), 3286-3295 (2016).</li><li>Goldmacher, V. S., Tinnel, N. L., Nelson, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+pinocytosis+in+lymphoid+cells+has+a+low+capacity.">Evidence that pinocytosis in lymphoid cells has a low capacity.</a> Journal of Cellular Biology. 102 (4), 1312-1319 (1986).</li></ol>

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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid hydrate</td>
			<td>Sigma Aldrich</td>
			<td>M-2933</td>
			<td>(MES); component of activation buffer</td>
		</tr>
		<tr>
			<td>50x2.5x1 mm magnets, Nickel (Ni-Cu-Ni) plated, grade N52, magnetized through 5mm (0.197&#34;) thickness</td>
			<td>K&#38;J Magnetics</td>
			<td>Custom</td>
			<td>Magnets used for the magnetic levitation device</td>
		</tr>
		<tr>
			<td>Capillary Tube Sealant (Critoseal)</td>
			<td>Leica Microsystems</td>
			<td>267620</td>
			<td>Used to cap the ends of the capillary tubes</td>
		</tr>
		<tr>
			<td>Centrifuge tube filters (Corning Costar Spin-X)</td>
			<td>Sigma Aldrich</td>
			<td>CLS8163</td>
			<td>Used to wash beads</td>
		</tr>
		<tr>
			<td>Compact Lab Jack</td>
			<td>Thorlabs</td>
			<td>LJ750</td>
			<td>Used for adjusting the magnetic levitation device</td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Solution for bead suspensions</td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma Aldrich</td>
			<td>E9508-100ML</td>
			<td>Used during a wash step for beads</td>
		</tr>
		<tr>
			<td>Fluorescent Plasma Membrane Stain (CellMask Green)</td>
			<td>Invitrogen</td>
			<td>C37608</td>
			<td>Used to stain Rh+ cells</td>
		</tr>
		<tr>
			<td>Gadoteridol Injection</td>
			<td>ProHance</td>
			<td>NDC 0270-1111-03</td>
			<td>Gadolinium (Gd3+); magnetic solution used to suspend cells</td>
		</tr>
		<tr>
			<td>HBSS++</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td>Solution for sample preparation</td>
		</tr>
		<tr>
			<td>Human C5b,6 complex</td>
			<td>Complement Technology, Inc</td>
			<td>A122</td>
			<td>Used to generate RBC Evs</td>
		</tr>
		<tr>
			<td>Human C7 protein</td>
			<td>Complement Technology, Inc</td>
			<td>A124</td>
			<td>Used to generate RBC Evs</td>
		</tr>
		<tr>
			<td>Human C8 protein</td>
			<td>Complement Technology, Inc</td>
			<td>A125</td>
			<td>Used to generate RBC Evs</td>
		</tr>
		<tr>
			<td>Human C9 protein</td>
			<td>Complement Technology, Inc</td>
			<td>A126</td>
			<td>Used to generate RBC Evs</td>
		</tr>
		<tr>
			<td>Mini Series Post Collar</td>
			<td>Thorlabs</td>
			<td>MSR2</td>
			<td>Used to secure magnetic levitation device to lab jacks</td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>E1769-10G</td>
			<td>(EDC); used in antibody coupling reaction</td>
		</tr>
		<tr>
			<td>Normal Rabbit IgG Control</td>
			<td>R&#38;D Systems</td>
			<td>AB-105-C</td>
			<td>Used to coat beads as a control condition</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (10X Solution, pH 7.4)</td>
			<td>Boston Bioproducts</td>
			<td>BM-220</td>
			<td>Component of coupling buffer, used for washing steps</td>
		</tr>
		<tr>
			<td>Polysorbate 20 (Tween 20)</td>
			<td>Sigma Aldrich</td>
			<td>P7949-500ML</td>
			<td>Component of activation buffer</td>
		</tr>
		<tr>
			<td>Polystyrene Carboxyl Polymer</td>
			<td>Bangs Laboratories</td>
			<td>PC06004</td>
			<td>Top density beads (1.05 g/mL), used for antibody coupling</td>
		</tr>
		<tr>
			<td>Rabbit RhD Polyclonal Antibody</td>
			<td>Invitrogen</td>
			<td>PA5-112694</td>
			<td>Used to coat beads for the dectection of Rh factor in red blood cells</td>
		</tr>
		<tr>
			<td>Research Grade Microscope</td>
			<td>Olympus</td>
			<td>Provis AX-70</td>
			<td>Microscoped used to mount magnetic levitation device and view levitating cells</td>
		</tr>
		<tr>
			<td>Rubber Dampening Feet</td>
			<td>Thorlabs</td>
			<td>RDF1</td>
			<td>Used to support the breadboard table</td>
		</tr>
		<tr>
			<td>Square Boro Tubing</td>
			<td>VitroTubes</td>
			<td>8100-050</td>
			<td>Capillary tube used for loading sample into Maglev</td>
		</tr>
		<tr>
			<td>Sulfo-NHS</td>
			<td>Thermoscientific</td>
			<td>24510</td>
			<td>Used in antibody coupling reaction</td>
		</tr>
		<tr>
			<td>Translational Stage</td>
			<td>Thorlabs</td>
			<td>PT1</td>
			<td>Used for focusing and for scanning capillary tube</td>
		</tr>
	</tbody>
</table>

quantification of cellular densities and antigenic properties using magnetic levitation

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。

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?...

Watch this Scientific Journal Video about Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation at JoVE.com

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

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。

使用磁悬浮定量细胞密度和抗原特性

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...

该技术允许分离各种细胞类型和仅基于密度进行各种过程的细胞，在短时间内几乎没有操作。这种方法的主要优点是它具有微创性，需要小体积，提供快速的结果，不依赖于经典的读数，并且不需要专业人员。该技术可用于检测多种疾病，例如贫血和败血症。首先使用一键式采血装置刺破恒河猴因子阳性献血者的手指，并在一毫升DPBS中收集10微升血液。在一毫升恒河猴因子阳性细胞的悬浮液中加入一微升荧光染料，对质膜进行荧光染色，并在37摄氏度下孵育15分钟。通过以5，600倍G旋转15秒来沉淀细胞，并使用一毫升DPBS洗涤沉淀三次。将沉淀重悬于一毫升HBSS中，加入钙和镁。使用一键式采血装置刺破恒河猴因子阴性献血者的手指，采集2微升血液。为了制备仅含有微珠的实验管，添加174微升HBSS与钙和镁，一微升IgG对照珠，一微升重珠，密度为每毫升1.2克，以及24微升500毫摩尔钆。制备含IgG的实验对照管，加入172微升HBSS含钙镁、1微升IgG对照珠、1微升重珠、1微升恒河猴因子阴性血液、1微升染色恒河猴因子阳性血液悬浮液、24微升500毫摩尔钆。制备样品实验管，加入172微升HBSS含钙和镁，1微升抗RhD包被珠，1微升重珠，1微升恒河猴因子阴性血液，1微升染色恒河猴因子阳性血液悬浮液，24微升500毫摩尔钆。设置磁悬浮装置。并将50微升的样品装入毛细管中，直到管子充满。用毛细管密封剂密封毛细管的末端，确保没有气泡。将毛细管放在顶部和底部磁铁之间的毛细管支架中，调整载物台和对焦以获得最佳观看效果。等待大约5到20分钟，让细胞和磁珠稳定在其磁平衡位置。使用该协议，使用磁悬浮装置根据悬浮高度分离血细胞。使用低密度和高密度珠子来提供密度悬浮高度和尺寸参考。多形核细胞悬浮在21毫摩尔钆离子浓度的红细胞上方。旧血细胞悬浮在60毫摩尔钆离子浓度下。将红细胞、多形核细胞和淋巴细胞根据其内在密度以40毫摩尔钆离子浓度分离。使用IgG和抗RhD抗体包被的珠子在红细胞上检测恒河猴因子抗原。IgG对照样品中未产生珠红细胞复合物，但检测到恒河猴因子阳性珠子捕获的荧光染色红细胞。抗RhB与恒河猴因子阳性细胞的结合在中心以未结合的珠子和阴性红细胞的间歇密度在中心产生珠细胞复合物。此外，通过观察发射的荧光证实了复合物的形成。涂有CR1和CD47抗体的高密度和低密度微球之间形成的B珠复合物表明存在红细胞衍生的细胞外囊泡。在不引入气泡的情况下盖上毛细血管是最具有挑战性的步骤，需要练习。这种方法可以为血液学提供见解，特别是专注于红细胞疾病。

Research

JoVE Journal

Biology

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

科研

生物学

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Quantifying Mixing using Magnetic Resonance Imaging

量化混合使用磁共振成像

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

细胞壁的弹性性能的原子力显微镜为基础的映射：在组织，细胞，亚细胞和决议

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

选择反应监测质谱法进行绝对定量蛋白质

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Cellular Redox Profiling Using High-content Microscopy

使用高含量显微镜的细胞氧化还原分析

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Fish Sperm Assessment Using Software and Cooling Devices

使用软件和冷却装置进行鱼类精子评估

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

使用前 Vivo 共聚焦显微镜可视化淋巴节点结构和细胞定位

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

低温样品加载到魔法角度旋转核磁共振光谱仪中，可保持细胞生存能力

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

使用单细胞荧光成像对菌际竞争进行量化

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...