我们想感谢资助的伍斯特理工学院化学与生物化学系。

1。转染细胞系转染法1 （龙沙Nucleofection为增加原代细胞的转染效率） <ol><li>细胞应该是最近通过的，并在转染前数期增长。洗净phophate细胞缓冲液（PBS）和收获细胞由胰蛋白酶。 </li><li>在1500 XG 5分钟，离心收集细胞。 </li><li>添加消毒盖玻片的6孔​​板。盖玻片，可增强细胞粘附涂层。广场1.5毫升培养基（在这种情况下贝科的改良Eagle培养基（DMEM），10％胎牛血清（FBS），）每孔和平衡在孵化器的媒体。 </li><li>收获细胞胰蛋白酶和悬浮在最小的体积的磷酸盐缓冲液（PBS）。 </li><li>计数细胞，离心再悬浮的正确金额，提供 5％〜5X10样品细胞。 </li><li>仔细的细胞重悬于100μL室温每个样品Nucleofector解决方案。 </li><li>联合收割机100μL的细胞悬液与5μg质粒DNA和样品转移到Nucleofector试管，小心不要包括气泡。 </li><li>选择适当Nucleofector的程序（这可能需要以前的测试，以确定最佳的转染效率与死亡率最低）。 </li><li> Nucleofector试管架含有悬浮细胞/ DNA插入试管，并开始选择的程序。 </li><li>完成后，取出试管，并添加〜500μL预平衡文化传媒（6孔板）。 </li><li>立即转移到6孔板终体积为1.5毫升，每孔样品。细胞应该是镀在任一混合种群或单独控制。 </li></ol>转染法2 （Qiagen公司Effectene转染细胞系） <ol><li>准备使用Qiagen公司Effectene试剂的转染复合物，由先加入每个质粒DNA样本的0.8微克至200μL欧共体缓冲区。 </li><li>每个样品中加入6.4μL，增强试剂，混合短暂弹管，为2分钟，在室温下孵育。 </li><li>每个样品中，混合加入20μLEffectene试剂弹管，15分钟在室温下孵育。 </li><li>在此孵化，准备转染细胞系的利益。最近传代细胞（&lt;80％汇合）洗一次在磷酸盐缓冲液（PBS）。在这种情况下加回1.5毫升新鲜的温暖和平衡增长中期（贝科改良Eagle培养基（DMEM），10％胎牛血清（FBS））。 </li><li>一旦转染复合物孵育15分钟，1.2毫升的媒体转移到每个样品。混合吹打，并立即传送〜700μL的配合，在6孔板每两个水井。加入滴加，旋流板，轻轻混匀。允许细胞转染至少3个小时（可能会随细胞类型）来完成。 </li><li>消毒盖玻片添加到一个新的6孔板。盖玻片，可增强细胞粘附涂层。 </li><li>转染细胞后，洗细胞两次，用PBS和收获最小的trysinization细胞，每孔加入约100μL胰蛋白酶溶液，简要搅拌充分大衣每口井的板，并立即吸胰蛋白酶。一旦细胞已经解除，加1.5毫升的新鲜传媒。 </li><li>此时，细胞混合或单独到无菌盖玻片的人口（控制）应镀。 </li><li>让细胞恢复了12个小时。 </li></ol> 2。细胞融合<ol><li>删除从细胞的培养基，用PBS洗两次。 </li><li>在这一点上，它是建议治疗与放线菌酮（100微克/毫升）的细胞，抑制蛋白质的合成。允许细胞孵育2小时，然后用PBS洗涤细胞两次。 </li><li>保险丝细胞暴露在室温为125秒，50％聚乙二醇1000（PEG 1000），在无血清DMEM溶液。 </li><li>用PBS清洗细胞，以消除聚乙二醇1000解决方案，并加入2 ml新鲜温暖和平衡的媒体。 </li><li>允许细胞孵育18 - 24小时。 </li></ol>荧光显微镜<ol><li>取出培养基，并在PBS洗细胞两次。 </li><li>加入1毫升4％多聚甲醛溶液（PBS）的每口井，修复细胞。轻轻搅拌15分钟。 </li><li>在PBS洗两次固定细胞。 </li><li>小心地从板盖玻片，灯芯多余的PBS。反转到显微镜玻片上装有安装介质的下降（90％甘油，100MM的Tris pH值7.5，2％的DABCO（1,4 - diazabicoyclo辛烷值）含有的DAPI（4&#39;,6 - diamidino - 2 -苯基吲哚）。 </li><li>从幻灯片中删除多余的安装介质，密封合作VER单用指甲油。 </li><li>通过共聚焦或epifluorescence显微镜可视化的细胞。 </li></ol> 3。代表性的成果图2显示了一个例子异核实验，使用的主要的成纤维细胞和H1299非小细胞肺癌细胞。在这个实验的兴趣蛋白（鸡贫血病毒VP3）epifluorescence显微镜观察显示本地化主要是细胞质的主要细胞类型，并在转化细胞类型的核本地化。作为一个融合（无论是绿色荧光蛋白的pEGFP - N1载体，Clontech公司）在主包皮成纤维细胞（PFF）或DsRed的H1299细胞（DsRed的N1载体，Clontech公司）的蛋白表达。控制样品涉及自我融合（前两行）显示多核细胞，在稳定状态下的本土化模式没有改变。这种变化，否则可能出现由于融合条件或syncitia形成的敏感性。异核体的融合（下排）证明GFP融合的主要细胞衍生的蛋白质和引进转化的细胞材料DsRed的融合蛋白的共定位核项目。显示的例子是一种比较罕见的只有两个单元，每个类型之一存在两个氟蛋白信号表明，异核体融合。除了检测本地化转换这些类型，核质穿梭活动，可以很容易被发现在两个细胞核的单荧光团的存在。这种类型的决心是明确的审查1:1细胞融合时，将取决于抑制翻译。 <img src="/files/ftp_upload/2488/2488fig1.jpg" alt="图1" /> 图1。流程图异核体使用主成纤维细胞和非小细胞肺癌细胞H1299的方法 。感兴趣的基因被克隆产生帧融合两个荧光蛋白基因之一。要么构造，然后瞬时转染细胞系，并允许恢复。异核体的形成是诱发进一步孵化与聚乙二醇（PEG）进行了简短的治疗。最后，感兴趣的蛋白质的亚细胞定位是使用各种形式的荧光显微镜观察。 <img src="/files/ftp_upload/2488/2488fig2.jpg" alt="图2" /> 图2。胞核贩运和鸡贫血病毒VP3蛋白的穿梭活动可视化的异核体的融合。首行:代表的图像，中间一排的自我融合H1299细胞表达DsRed的- VP3:小学包皮成纤维细胞的PEG融合后表达GFP - VP3（PFF）代表epifluorescence 图像底部行 :PFF和H1299细胞的异核体的融合。黄色表示的绿色荧光蛋白DsRed的信号的共定位。细胞成像使用徕卡AF6000E荧光显微镜和Leica成像软件。

<ol>
	<li>Belaya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLIPing+heterokaryons+to+analyze+nucleo-cytoplasmic+shuttling+of+yeast+proteins.">FLIPing heterokaryons to analyze nucleo-cytoplasmic shuttling of yeast proteins.</a> RNA. 12, 921-930 (2006).</li><li>Bhaumik, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+target+of+a+transcriptional+activator+revealed+by+fluorescence+resonance+energy+transfer.">In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer.</a> Genes Dev. 18, 333-343 (2004).</li><li>Cohen, H. R., Panning, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XIST+RNA+exhibits+nuclear+retention+and+exhibits+reduced+association+with+the+export+factor+TAP/NXF1.">XIST RNA exhibits nuclear retention and exhibits reduced association with the export factor TAP/NXF1.</a> Chromosoma. 116, 373-383 (2007).</li><li>Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dapper1+is+a+nucleoplasmic+shuttling+protein+that+negatively+modulates+Wnt+signaling+in+the+nucleus.">Dapper1 is a nucleoplasmic shuttling protein that negatively modulates Wnt signaling in the nucleus.</a> J. Biol. Chem. 51, 35679-3588 (2008).</li><li>Grespin, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+Hormone+Receptor+&#945;1+Follows+a+Cooperative+CRM1/Calreticulin-mediated+Nuclear+Export+Pathway.">Thyroid Hormone Receptor &#945;1 Follows a Cooperative CRM1/Calreticulin-mediated Nuclear Export Pathway.</a> J. Biol. Chem. 283, 25576-25588 (2008).</li><li>Heilman, D. W., Teodoro, J. G., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptin+Nucleocytoplasmic+Shuttling+Is+Required+for+Cell+Type-Specific+Localization,+Apoptosis,+and+Recruitment+of+the+Anaphase-Promoting+Complex/Cyclosome+to+PML+Bodies.">Apoptin Nucleocytoplasmic Shuttling Is Required for Cell Type-Specific Localization, Apoptosis, and Recruitment of the Anaphase-Promoting Complex/Cyclosome to PML Bodies.</a> J. Virology. 80, 7535-7545 (2006).</li><li>Jeffries, S., Capobianco, 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=Neoplastic+transformation+by+Notch+requires+nuclear+localization.">Neoplastic transformation by Notch requires nuclear localization.</a> Mol Cell Biol. 20, 3928-3941 (2000).</li><li>Keaton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleocytoplasmic+trafficking+of+G2/M+regulators+in+yeast.">Nucleocytoplasmic trafficking of G2/M regulators in yeast.</a> Mol. Biol. Cell. 19, 4006-4018 (2008).</li><li>Ling, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear-Cytoplasmic+Shuttling+Is+Not+Required+for+the+Epstein-Barr+Virus+EBNA-LP+Transcriptional+Coactivation+Function.">Nuclear-Cytoplasmic Shuttling Is Not Required for the Epstein-Barr Virus EBNA-LP Transcriptional Coactivation Function.</a> J. Virology. 83, 7109-7116 (2009).</li><li>Lin, X. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RXR&#945;+acts+as+a+carrier+for+TR3+nuclear+export+in+a+9-cis+retinoic+acid-dependent+manner+in+gastric+cancer+cells.">RXR&#945; acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells.</a> J. Cell Science. 117, 5609-5621 (2004).</li><li>Vissinga, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Export+of+NBN+Is+Required+for+Normal+Cellular+Responses+to+Radiation.">Nuclear Export of NBN Is Required for Normal Cellular Responses to Radiation.</a> Mol Cell Biol. 29, 1000-1006 (2009).</li><li>Zimmerman, R. S., Rosenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+p19Arf+localization+accompany+apoptotic+crisis+during+pre-B-cell+transformation+by+Abelson+murine+leukemia+virus.">Changes in p19Arf localization accompany apoptotic crisis during pre-B-cell transformation by Abelson murine leukemia virus.</a> J. Virol. 82, 8383-8391 (2008).</li></ol>

这里介绍的异核协议提供了一个高效和容易解释的方法对细胞的特定类型的本地化行为，以及核质穿梭活动表征。此方法采用荧光分析的灵敏度，以及图像的活细胞和执行蛋白质动力学的研究能力的优势。该技术是很容易适应许多不同类型的细胞，是适合生活和固定细胞成像。例如，倒置荧光显微镜可用于实时成像和时间的推移视频采集。相比之下，安装细胞可用于epifluorescence显微镜无论是稳态本地化或更详细的共焦成像离散本地化的决心。此外，如荧光漂白后恢复（FRAP），或在漂白荧光丢失（FLIP）的技术可用于确定本地化动力学 1 。协议的替代荧光团的法团，将允许的蛋白质相互作用的实验荧光共振能量转移（FRET） 将在演唱会2进行。 leptomycin B或显性负，如蜂窝贩运的各种抑制剂冉GTP酶的结构，可以用来建立对特定进口或出口的途径5,6,9的依赖。 <ul><li>在某些实验涉及穿梭活动中，必须小心，以避免由于新的蛋白质合成的本地化效果。在这种情况下，转换酶抑制剂或有限的时间点必须使用。然而，由于翻译抑制的文物仍然是一个可能性。含有不同的细胞类型的细胞融合是最想要的穿梭和本地化的行为明确的解释。优化细胞的数量和融合条件（时机和浓度）可能是必要的，以促进这种良好的1:1融合事件。 </li><li>重要的是要注意，在协议中的某些步骤的优化，将需要使用特定的细胞类型而定。在实验过程中，如融合效率，转染效率，融合后的生存能力方面会有很大的改变之间的细胞类型。尤其是主要的细胞类型可以是具有挑战性的管理，由于复杂的文化条件和转染效率低。龙沙Nucleofector设备，如技术的使用可以使细胞的快速处理，以及转染效率显着提高。此外，电的方法，使悬浮液中的细胞融合的后续步骤，便于转移。 </li></ul>

<table><tbody><tr><td>Effectene reagent</td><td>Qiagen</td><td>301425</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P5493</td><td></td></tr><tr><td>Trypsin</td><td>Fisher Scientific</td><td>SH3023602</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Fisher Scientific</td><td>SH30243FS</td><td>High glucose</td></tr><tr><td>paraformaldehyde</td><td>Fisher Scientific</td><td>O4042-500</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>Sigma-Aldrich</td><td>D9542-10MG</td><td></td></tr><tr><td>Hyclone Serum (fetal bovine)</td><td>Thermo Fisher Scientific, Inc.</td><td>SH3008803HI</td><td></td></tr><tr><td>Falcon 6-well plates</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>Polyethylene glycol 1000</td><td>Fisher Scientific</td><td>528875-25GM</td><td></td></tr><tr><td>Cover slips</td><td>Fisher Scientific</td><td>12-545-85</td><td></td></tr><tr><td>DABCO</td><td>Sigma-Aldrich</td><td>D2522-25G</td><td></td></tr><tr><td>Nucleofector HDF kit</td><td>Lonza Inc.</td><td>VPD-1001</td><td></td></tr></tbody></table>

heterokaryon technique for analysis of cell type-specific localization

大量的蛋白质，是受细胞内贩卖或nucleocytolasmic穿梭。这些蛋白质显示了包括核进口/出口RNA和蛋白质，转录调控和细胞凋亡的细胞功能的多样化。有趣的是，主要包括细胞分裂，分化和改造的细胞重组，往往涉及这类活动 3,4,8,10 。这些蛋白质和各自的监管机制的详细研究是具有挑战性的，这些本地化的变化可以难以捉摸的刺激，和运动本身可以是相当动态的，难以跟踪。涉及细胞癌变的研究，例如，继续受益于理解的途径和蛋白质的活动之间的正常原代细胞和转化细胞6,7,11,12不同。由于许多蛋白质​​在改变本地化改造或作为一个转型的结果，有效地描述这些蛋白质的方法和途径其所参与的立场，以改善癌变和靶向药物开辟新的领域的理解。 这里我们提出一个分析蛋白质贩运的方法，小学和转化哺乳动物细胞之间的穿梭活动。这种方法结合荧光显微镜的异核体融合的生成提供了一个灵活的协议，可用于检测的稳态或动态的蛋白质本地化。正如图1所示，两个独立的细胞类型，瞬时转染轴承氟蛋白基因连接到感兴趣的基因质粒结构。表达后，细胞融合使用聚乙二醇，蛋白质本地化可能使用多种方法进行成像。这里介绍的协议可能会增加专业技术的一个根本途径。

Watch this Scientific Journal Video about Heterokaryon Technique for Analysis of Cell Type-specific Localization at JoVE.com

Heterokaryon Technique for Analysis of Cell Type-specific Localization

一个细胞类型特异性的蛋白定位和穿梭核质特性的灵活和有效的方法是描述。这异核体的方法是使用荧光标记的融合蛋白细胞融合后图像蛋白质本地化。该协议是经得起稳态本地化或活细胞成像的基础上更具活力的测定。

异核技术的细胞类型特异性定位分析

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

heterokaryon-technique-for-analysis-of-cell-type-specific-localization

Research

JoVE Journal

Biology

16.4K 次观看.  Worcester Polytechnic Institute- WPI. 一个细胞类型特异性的蛋白定位和穿梭核质特性的灵活和有效的方法是描述。这异核体的方法是使用荧光标记的融合蛋白细胞融合后图像蛋白质本地化。该协议是经得起稳态本地化或活细胞成像的基础上更具活力的测定。

视频: Heterokaryon Technique for Analysis of Cell Type-specific Localization

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

科研

遗传学

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

细胞谱系分析和基因功能研究使用双点MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

发育生物学

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

生物学

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

神经科学

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

标记的细胞核从亲和力为基础的隔离<em&gt;果蝇</em&gt;组织的基因表达分析

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

活体哺乳动物细胞器中单膜蛋白的多色定位显微术

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

An Immunolabeling Technique to Visualize the Subcellular Localization of Proteins

Source: Aishwarya, R., et al. <a href="https://app.jove.com/v/64085">Visualizing Subcellular Localization of a Protein in the Heart Using Quantum Dots-Mediated Immuno-Labeling Followed by Transmission Electron Microscopy.</a> J. Vis. Exp. (2022).
This video demonstrates a technique for the immunolabeling of sigma-1 receptors in a murine heart tissue section using quantum dots. The heart tissue is fixed, osmicated, stained, dehydrated, embedded in resin, and then sectioned. The sections are then treated with antibodies and labeled with quantum dots to demarcate the subcellular localization of Sigma-1 receptors using electron microscopy.

一种可视化蛋白质亚细胞定位的免疫标记技术

1. Animal preparation

	Anesthetize the mice using 3% isoflurane. Open the chest by making a horizontal incision in the region above the middle abdomen ...