这项工作是由老鹰创新奖（联谊订购老鹰）和由梅奥基金会的支持。

**本视频协议是基于相关出版物1：本机的蛋白质相互作用的多蛋白复合物和流式细胞仪的高灵敏度检测和定量分析。亚当G. Schrum，黛安娜吉尔，伊莱恩体育Dopfer，大卫L WIEST，劳伦斯A. Turka，沃尔夫冈西澳Schamel，埃德 - 帕尔默。 STKE 2007年“科学”（389）：PL2，6月5日，2007年，[作者：10.1126/stke.3892007pl2] <a href="http://stke.sciencemag.org/cgi/content/abstract/2007/389/pl2">请点击这里查看本出版物</a> 。 开始之前，请准备以下的水库存解决方案： <table><tbody><tr><td> MES的耦合缓冲： </td><td> 储存在25 ° C </td></tr><tr><td> MES，pH值6.0 </td><td> 50毫米</td></tr><tr><td> EDTA </td><td> 1毫米</td></tr><tr><td> 淬火/阻止/存储缓冲区（QBS）： </td><td> 保存在4 ° C </td></tr><tr><td>牛血清白蛋白</td><td> 1％ </td></tr><tr><td>叠氮化钠</td><td> 0.02％ </td></tr><tr><td> PBS，pH值7.4 </td><td> 1X </td></tr><tr><td> FCM的缓冲区： </td><td> 保存在4 ° C </td></tr><tr><td>三，pH值7.4 </td><td> 50毫米</td></tr><tr><td>氯化钠</td><td> 100毫米</td></tr><tr><td>叠氮化钠</td><td> 0.02％ </td></tr><tr><td>胎牛血清</td><td> 5％ </td></tr><tr><td> PBS，pH值7.4 </td><td> 1X </td></tr></tbody></table>立即准备使用前： <table><tbody><tr><td> EDAC - MES的： </td><td>在MES耦合缓冲液溶解50毫克/毫升EDAC的粉</td></tr><tr><td> 毛地黄皂苷溶液（2％W / V）： </td><td>卫生署2 O溶解毛地黄皂苷粉末加热到95℃，5分钟，然后在冰上冷却</td></tr><tr><td> 裂解缓冲液： </td><td></td></tr><tr><td>三，pH值7.4 </td><td> 50毫米</td></tr><tr><td>氯化钠</td><td> 150毫米</td></tr><tr><td>毛地黄皂苷的解决方案</td><td> 1％ </td></tr><tr><td>蛋白酶抑制剂</td><td> 1X </td></tr><tr><td> 置于冰上 </td><td></td></tr></tbody></table> 1。耦合珠单抗<ol><li>从购买股票的确定珠的浓度稀释在PBS和一个hemacytometer计数。 1:10000稀释的珠子开始计数。 </li><li>移液器18 × 10 6到1.5 ml离心管珠。 </li><li>洗磁珠在0.5至1.0毫升MES偶联缓冲2到3倍，在20,000克3分钟，在25 ° C离心后每洗。 </li><li> 50μLMES偶联缓冲液重悬珠。 </li><li>加入20μL新鲜配制的EDAC - MES激活的羧基上的珠子。 </li><li>在25 ° C通过手动移液器上下轻轻搅拌15分钟。 </li><li>洗净激活珠2至3倍，在0.5至1.0毫升PBS离心20,000克，3分钟后在25 °每个洗的C。 </li><li>在50μL的PBS重悬激活珠。 </li><li>加入50μL的知识产权人与生物圈计划（股权集中在0.2-1.0毫克/毫升）激活珠的解决方案。 </li><li> 3至4小时在25 ° C的混合放在振动筛管。摇到足以防止沉淀在试管底部珠。 </li><li>洗净人与生物圈计划加上珠在0.5至1.0毫升PBS的2至3倍，离心3分钟，在20000克在25 ° C后，每次冲洗。 </li><li>重悬于100缓冲液QBS珠。这些都可以存储在4 ° C </li><li>暂停的珠子，在PBS稀释1:200-1:10,000与hemacytometer和计数。浓度必须测量每个制备批，可精​​确控制，使每个IP或下拉实验中使用的珠数。 </li></ol> 2。后核裂解液的制备及IP 裂解法和最佳的裂解条件，将取决于细胞类型，并正在研究蛋白质相互作用。的协议存在，并且可以为您的应用程序进行修改。 <ol><li>裂解20 × 10 6&#39;小&#39;细胞，如淋巴细胞，或5 × 10 6“大”的细胞，如巨噬细胞或肿瘤细胞，在100μL新鲜的1.5 ml离心管放在冰上20分钟的裂解缓冲。需要规模的溶解量。 </li><li>为了去除细胞核和不溶性的细胞碎片，离心20,000克的裂解液在4 ° C 10分钟保持上清，弃去沉淀。 </li><li>添加0.5 × 10 5〜2.5 × 10 5人与生物圈计划加上珠澄清裂解液，轻轻吹打混合。我们使用的体积最小执行一个单一的IP为5μL。 </li><li>广场上的一个垂直旋转的轮子4小时，在寒冷的房间里过夜。设置足够的速度，以防止产生沉淀珠。量低IPS液体留在试管底部，在旋转过程中是可以接受的。 </li></ol> 3。磁珠捕获蛋白质的探讨与荧光标记的抗体<ol><li>洗净的IP珠在0.2至1.0毫升冰冷的FCM缓冲区，两次，离心20,000克在4 ° 3分钟后，每次冲洗的彗星。 </li><li>重悬于100μL的FCM缓冲区和分装20μL到每个五年或以上的1.5 ml离心管或烟囱底板井珠。 </li><li>样品加入荧光标记的单克隆抗体。执行染色的FCM根据供应商的指示，或根据经验确定的浓度的。作为一个起点，每管或添加0.2至1μL股票抗体溶液（0.2-1.0毫克/毫升），孵育40分钟冰。 </li><li>洗净探讨在1.5毫升管珠两次为1.0 mL冰冷的FCM缓冲区，在20000克离心4 ° C为3分钟后，每次冲洗。每次清洗后轻轻地取出上清液，注意不要打扰珠。烟囱底板，用0.2毫升冰冷的FCM缓冲区，两次，离心5分钟，在4千克° C后每洗。多通道移液器这一步是非常有用的。若要从烟囱底板珠上清，“甩尾”的板一次，然后，同时仍然倒挂式，印迹边缘井滴水。在每口井的残留量，含珠，将约20μL。 </li><li>重悬在每个样品200μL的FCM缓冲区的珠子。转移标记流式细胞仪管。样品现已FCM的准备。 </li></ol> 4。 FCM的收购在本议定书中所描述的慢性粒细胞白血病的珠子直径3至5微米，直径大约一半的一个静态的小鼠淋巴细胞。因此，它可能需要手动增加的前向散射（FSC）的放大器增益和侧散射（SSC），电压为珠事件的人口登记规模。单个IP珠子，应该形成一个紧密集群的单身人口。设置和门，应进行调整，以排除珠双峰和碎片。 <ol><li>之前运行的珠片或珠标准，除去周围一些流式细胞仪的进样液滴遏制套。允许鞘液样本之间的点滴，因为这将明确的入口和防止珠被结转从一个样品到另一个。 </li><li>使用未标记的珠子，阴性对照的荧光，和彩虹校准颗粒（垃圾站），阳性对照的荧光，对设置进行调整，使双方的消极和积极的荧光极端同时出现在日志规模的X轴。这保证了实验样品的荧光，也将在规模。请注意，垃圾收集站，比CML珠小，因此，FSC和SSC参数，以及门的位置，可能需要暂时予以改变，样品。 </li><li>与垃圾收集站的校准完成后，返回到未标记的珠设置。如果只有每一个样品的荧光探针，无荧光的补偿是必要的。在一般情况下，我们探头与每一个单一的荧光标记单克隆抗体流式细胞仪样品的多蛋白复合物，和我们研究了多种互动的合作伙伴，通过与具体的各亚基的单克隆抗体染色平行珠样品。 </li><li>采集和保存数据文件荧光。分析然后可以使用流量，如流式细胞仪软件CellQuest，FlowJo，或cflow的。 </li></ol> 5。代表性的成果<img src="/files/ftp_upload/2066/2066fig1.jpg" alt="图1" /> 图1。 TCR/CD3多蛋白复合物的知识产权的FCM，T细胞的小鼠品系的BALB / C溶解在1％毛地黄皂苷，和裂解受到IP - FCM使用抗CD3ε珠。捕获复合物含有大量的T细胞受体-β（紫色区域），并联合相关CD3 -ε（绿色），但接近其他蛋白质，如Thy1.2的背景水平（不相关的免疫球蛋白探头，粉红色的跟踪定义）， CD45，或H - 2K（D）（棕色，橙色和蓝色的痕迹，分别）。类似先前公布的结果1-6。

<ol>
	<li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+detection+and+quantitative+analysis+of+native+protein-protein+interactions+and+multiprotein+complexes+by+flow+cytometry.">High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.</a> Sci STKE. , pl2-pl2 (2007).</li><li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+multiprotein+complexes+by+flow+cytometry.">Visualization of multiprotein complexes by flow cytometry.</a> Curr Protoc Immunol. Chapter 5, 9-9 (2009).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+division+and+death+are+segregated+by+mutation+of+TCRbeta+chain+constant+domains.">T cell division and death are segregated by mutation of TCRbeta chain constant domains.</a> Immunity. 21, 515-526 (2004).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+T+Cell+receptor+signals+determine+CD8++memory+versus+effector+development.">Different T Cell receptor signals determine CD8+ memory versus effector development.</a> Science. 323, 502-505 (2009).</li><li>Treves, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctate+is+a+key+element+in+calcium+entry+induced+by+activation+of+InsP3+receptors+and/or+calcium+store+depletion.">Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion.</a> J Cell Biol. 166, 537-548 (2004).</li><li>Gil, D., Schrum, A. G., Alarcon, B., Palmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+engagement+by+peptide-MHC+ligands+induces+a+conformational+change+in+the+CD3+complex+of+thymocytes.">T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 complex of thymocytes.</a> J Exp Med. 201, 517-522 (2005).</li></ol>

有关蛋白质相互作用的信息是高度相关的许多细胞过程，如信号转导，谱系成熟，细胞周期的进展，和凋亡级联的分析。 IP - FCM提供了快速，定量和敏感的方法，研究蛋白质的相互作用，并定义多蛋白复合物的成员在他们的天然构象。珠可以加上和培养细胞裂解液在一天之内，可以探测和分析第二天。一个96孔板格式允许对大量样品分析的数字，在同一时间提供高效的数据收集统计或筛选的目的。使用荧光珠标准，每个珠子捕获的蛋白质的数量可以估算的。源材料是很少需要捕捉和检测，使有限的样品和稀缺的分析物仍然可以为多个交互分析。虽然不需要IP - FCM的基因工程技术，抗原表位标记，变性，或在体外混合蛋白在非生理性的环境，它可以加上这些和其他技术，使得它与适用性的宝贵和访问工具许多生物系统。 故障排除： 许多IP - FCM的实验产生有用的蛋白质相互作用数据，他们首次尝试。然而，IP - FCM的优化可以提高抗体共轭珠，蛋白质复合体捕获，荧光探针结合。 可通过直接探测反免疫球蛋白抗体偶联珠IP抗体共轭的效率。如果效率低，增加在偶联反应的抗体浓度，可以让更多的IP抗体附加到每个珠子。这可以提高IP珠一批具有约束力的能力，增强型捕捉和分析物的检测。其他主要的胺（如三，牛血清白蛋白），其中包含分子不应该存在的偶联反应过程中，因为这些都可以与单克隆抗体的竞争珠附着在单克隆抗体低耦合的结果。如果共轭珠单抗证明问题等原因，珠，而不是可以被耦合到亲和素/链霉亲，生物素化抗体可以随后非共价键结合和immunprecipitation使用。 尽管良好的抗体共轭，初步检测珠相关的荧光可能偏低。第一，复杂的捕捉本身可能是低的。由于IP - FCM取决于分析物的浓度，增加单位的溶解量裂解细胞的数量，可提高分析物的捕获和检测。此外，捕捉可能是通过减少孵育裂解液，分配捕获复合物横跨珠少，增加每珠平均荧光探测的结果IP珠增强。其次，它是可能的探头抗体捕获复合物的访问是立体immunoprecipitating抗体阻碍。在这种情况下，问题可以得到解决，使用不同的抗体捕获和/或探针。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>carboxylate-modified polystyrene latex (CML) beads (5mm surfactant-free)</td>
<td>Reagent</td>
<td>Interfacial Dynamics Corporation/Molecular Probes</td>
<td>2-5000</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>Rainbow Calibration Particles</td>
<td>Reagent</td>
<td>Spherotech, Inc.</td>
<td>RCP-30-5A</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>2-[N-Morpholino] ethanesulfonic acid (MES)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M-5287</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3-(3-dimethylaminopropl) carbodiimide HCl (EDAC)</td>
<td>Reagent</td>
<td>Pierce Sigma</td>
<td>22980 E-6383</td>
<td>Store at -20&deg;C under desiccating conditions.</td>
</tr>
<tr>
<td>Protease Inhibitor General Use Cocktail, containing AEBSF, Bestatin, Aprotinin, EDTA, E-64, and Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-2714</td>
<td> Prepare 100x stock solution in H2O. Aliquot and store at -20&deg;C. Handle with gloves.</td>
</tr>
<tr>
<td>PCR plates, 96-well 0.2mL, natural, tall chimney</td>
<td>Reagent</td>
<td>Fisher</td>
<td>08-408-230</td>
<td>For use in place of microcentrifuge tubes during FCM staining</td>
</tr>		</tbody>
		</table>
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ip-fcm:  immunoprecipitation detected by flow cytometry

通过流式细胞仪检测免疫沉淀（IP，FCM）是一种用于检测和定量蛋白质相互作用的有效方法。基本原则延伸夹心ELISA法，其中捕获的主要分析物可以检测到身体内多蛋白复合物相关的其他分子一起。该过程涉及与immunoprecipitating单克隆抗体（mAb）的具体利益的蛋白质，细胞裂解液中培育这些珠子，捕获的蛋白复合物与荧光标记的探针探测，并通过流式细胞仪分析珠相关的荧光聚苯乙烯乳胶微球的共价偶联。 IP - FCM是极其敏感的，允许在本机（非变性）状态的蛋白质分析，并服从或者半定量或定量分析。 IP - FCM需要作为额外的好处，没有基因工程或专门的设备，流式细胞仪以外，它可以很容易地适应高通量应用。

Watch this Scientific Journal Video about IP-FCM:  Immunoprecipitation Detected by Flow Cytometry at JoVE.com

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

IP FCM的方法是，允许一个敏感的，健壮的，原生的蛋白质 - 蛋白质相互作用的生化评估，而无需遗传工程或大样本。

IP - FCM：免疫沉淀，流式细胞仪检测

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

ip-fcm-immunoprecipitation-detected-by-flow-cytometry

Research

JoVE Journal

Biology

17.3K 次观看.  Mayo Clinic. IP FCM的方法是，允许一个敏感的，健壮的，原生的蛋白质 - 蛋白质相互作用的生化评估，而无需遗传工程或大样本。

视频: IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

抗体分析的荧光素酶免疫沉淀系统（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

科研

生物学

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED热流量 - 结合光遗传学与流式细胞仪

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

流式细胞仪检测盘基柄柄Macropinocytosis 的高通量

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...