Name: preparation of cd4+ t cells for analysis of gd3 and gd2 ganglioside membrane expression by microscopy
Uploaded: 2016-11-08T13:00:00.000+00:00
Duration: 10 min
Description: Watch this Scientific Journal Video about Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy at JoVE.com

We are grateful to Dr. Jos&#233; Luis Daniotti for comments. We thank the assistance to Dr. J. Arturo Pimentel and the Laboratorio Nacional de Microscop&#236;a Avanzada-UNAM for acquisition of confocal images. I.M.-D. and T.M.V.C. were supported by grants 157634 and 253596 from Consejo Nacional de Ciencia y Tecnolog&#236;a (CONACYT), Sociedad Latinoamericana de Glicobiolog&#236;a, A.C. T.M.V.-C. is recipient of a scholarship (245192) from CONACYT. We also thank the support of the Red Tem&#225;tica Glicociencia en Salud - CONACYT (253596).

与知情同意和炫酷德Investigaciòn连接DinámicaCelular-自治大学德尔斯卡德莫雷洛斯生物伦理委员会批准获得从健康男性献血者外周血。 1.分离人幼稚CD4 + T细胞活化<ol><li>收集2毫升从通过知情同意健康人供体衍生的外周血，并用2毫升无菌PBS-EDTA的稀（1×磷酸盐缓冲盐水（PBS），2毫摩尔EDTA，pH 7.4）中。关于3毫升蔗糖溶液的顶端缓慢加入稀释的血液与1.077密度如前所述8。 注意:所引起的密度差在离心分离期间微分迁移将导致红细胞完全沉淀和密度较小的单核细胞层将形成以上。加入2ml外周血将purificati后呈现约0.5×10 6幼稚CD4 + T细胞在步骤。 </li><li>在在21℃（使用转子用于与为30mm固定角度圆底管），并且没有断点250 XG离心30分钟至产生外周血单核细胞（PBMC）的梯度。离心后，将PBMC中可以清楚地观察到，为白色环。收集用移液器将PBMC和转移到无菌管中，洗涤。 </li><li>洗涤三次后，在250×g离心10分钟，PBS-EDTA溶液进行幼稚CD4 + T细胞的纯化。排序方法或几种类型的纯化试剂盒可用于该目的。优选地，使用&gt; 1×10 7个PBMC中纯化。 <ol><li>通过用磁珠阴性选择纯化幼稚CD4 + T细胞。用抗CD45RO，CD8，CD14，CD15，CD16，CD19，CD25，CD34，CD36，CD56，CD123，抗TCRγ/δ，抗HLA-DR，和CD235a（血型糖蛋白A）的抗体进行阴性选择。由至f确定的CD4 + CD45RA +细胞评估纯度的百分比低流式细胞仪。 注意:任选地，进行到PBMC中的孵育在37℃和5％的CO 2与在10ml补充的培养基的1×10 6个细胞/ ml的密度，以促进单核细胞粘附，并继续与幼稚CD4 + T细胞纯化过夜。从外周血幼稚CD4 + T细胞的有效回收很大程度上取决于净化系统。 </li></ol></li><li>通过加入每PBS的孔250微升含5微克/ ml抗CD3单克隆抗体制备的24孔细胞培养板中，并在37°C孵育至少2小时。 注:96孔板可以用来当使用从幼稚CD4 + T细胞数量较少（ 例如，2×10 4 -1×10 5）。 </li><li>除去从24孔或96孔板的抗CD3抗体的稀释和洗涤用无菌1×PBS洗涤两次板。 </li><li>用稀释纯度&gt; 95％的幼稚CD4 + T细胞（CD4 +CD45RA +）以补充有胎牛血清的3％的1×10 6个细胞/ ml，在先进的RPMI 1640培养基中的密度（或RPMI补充有血清的10％），2mM谷氨酰胺和抗生素（1 100U / ml青霉素，1 100μg/ ml链霉素）。 注意:如果神经节苷脂本地化外周血单个核细胞中要求，遵循同样的协议进行稀释和刺激，如下所述。 </li><li>加入500微升细胞稀释的抗CD3包被的孔，并在24孔板非涂布孔中（对照细胞）。备选地，加入100微升细胞稀释到96孔板的孔中。 </li><li>通过加入抗CD28抗体的1微克/毫升完成幼稚的 CD4 + T细胞在抗CD3包被的孔的刺激条件。 </li><li>保持静止的 CD4 + T细胞在非包被的孔控制在不加抗CD28抗体。孵育0静止和活化的CD4 + T细胞，以72小时的37℃的标准条件下C和5％ 的 CO 2。 </li><li>为了验证一个适当的活化静息流式细胞仪检测CD69早期活化标记（16小时激活后）或CD25晚期活化标志（48小时后激活）的表达，评估和激活，在37℃下培养幼稚CD4 + T细胞和5％的CO 2在不存在或抗CD3 / CD28抗体9,10的存在。 </li></ol> 2.休息的坚持和激活幼稚的CD4 + T细胞<ol><li>消毒通过高压和紫外线照射12毫米圆形盖玻片至少15分钟。 </li><li>地方盖玻片在无菌的24孔细胞培养板。 注意:对于具有小于1×10 5细胞培养物是优选使用滑动室有适于小孔，以防止细胞分散在盖玻片。 </li><li>涂层盖玻片（或滑动室）用200μl聚L-赖氨酸（MW 150-300千道尔顿）的0.1％（重量/体积）溶液，并孵育在r 5分钟OOM温度（RT），取出，晾干，在室温至少1小时。 </li><li>仔细混合并收集来自24孔板的休息和激活幼稚的 CD4 + T细胞。计数细胞，并在必要时用新鲜补充的培养基调整至1×10 5个细胞转移到聚-L-赖氨酸涂覆的盖玻片。孵育所述细胞在37℃下至少6小时和5％的CO 2。 </li></ol> 3.收集和休息的固定和激活幼稚的CD4 + T细胞<ol><li>小心地从培养的休息和激活幼稚的 CD4 + T细胞的培养基。 </li><li>添加RT无菌PBS500μl的慢。 注意:从井溶液仔细添加和去除防止细胞的分离从盖玻片。 </li><li>弃去PBS，并添加另外500微升PBS彻底清除培养基。 </li><li>加入500微升过滤4％多聚甲醛（FILT修复细胞配给移除可与显微镜分析干扰），并在RT（优选的）或在4℃过夜温育30分钟微粒。 注意:多聚甲醛是一种有毒的挥发性化合物。它被称为是一个人类致癌物。用适当的安全协议，请谨慎使用。 </li><li>除去固定剂，并在室温用PBS洗涤至少两次。 </li><li>继续进行下一步骤，或保持板孔用500μl的PBS在4℃下数天，直至染色。 注:在此过程中不育有助于避免微生物的生长。 </li></ol> 4.染色，安装和可视化激光共聚焦显微镜<ol><li>从孔中取出PBS中。加入500微升冷封闭缓冲液（1×PBS，0.5％的标准级牛血清白蛋白，1％胎牛血清pH 7.4）中。孵育在冰上20分钟。 </li><li>小心地取出封闭液，添加的主要抗体（抗神经节苷脂GD3克隆R24，GD2克隆14G2a）PE标记的抗CD25和APC缀合的抗TCR和同种型在封闭缓冲液以2.5微克/毫升稀释的控制。 </li><li>在冰上孵育2小时或过夜，在4℃。 注:缓慢轨道搅动是优选的。 </li><li>小心取出抗体和缓慢加入500微升的PBS。重复两次。 </li><li>添加二次抗体在封闭缓冲液以0.1微克/毫升稀释（为R24抗GD3，的Alexa Fluor为14G2a抗GD2 488缀合的或的Alexa Fluor 647缀合的抗IgG2a的FITC缀合的抗IgG3的）。 </li><li>孵育在黑暗冰1小时无晃动。 </li><li>在PBS中洗涤三次如4.4所述）。保持有足够的PBS的孔中，以避免样品脱水。 </li><li>添加赫斯特333258染色在PBS中稀释至0.1微克/毫升。 </li><li>孵育15分钟，在室温和删除。在PBS中洗涤三次。 </li><li>放置在纸巾上的幻灯片，并添加20微升安装溶液（在PBS中的50％的甘油）或商业安装溶液s到避免褪色和样品淬火。 </li><li>小心地拿起用细镊子盖玻片，并将其放置在安装的解决方案。确保在细胞附着在盖玻片的侧面与所述溶液接触。密封指甲油盖玻片并用荧光或共聚焦显微镜分析。 </li></ol> 5.隔离的RNA <ol><li>制备抗CD3抗体（5微克/毫升）包被的96孔板上。孵育4×10 4个幼稚CD4 + T细胞/孔在100μl补充培养基与抗CD28抗体（1μg/ ml的）来完成的刺激。在37℃和5％CO 2的0至72小时。 </li><li>不同的激活后次后放置细胞成离心管中。 </li><li>在250 XG添加的PBS 500微升，离心在RT 5分钟。 </li><li>除去上清液，加入1ml Trizol试剂。 </li><li>在室温下孵育5分钟，并保持样品在-8076℃持续至少24小时。在需要时与RNA分离进行。 注意:在-80℃下离开样品至少24小时提高RNA的产率。此外，利用手的手套是必不可少由RNA酶，以避免RNA降解。 </li><li>除霜温育的样品在37℃水浴中2分钟。 </li><li>加入200μl氯仿，用手30秒（以避免RNA侵略性混合涡旋）剧烈震荡。孵育5分钟，在室温。 </li><li>在4℃下12000 xg离心离心15分钟。 </li><li>回收水相到一个新的离心管中。 </li><li>加入500微升异丙醇并倒转试管三次。 </li><li>孵育在室温10分钟，离心10分钟以12,000 xg离心在4℃下。 </li><li>丢弃由倒置上清。 </li><li>以12,000 xg离心加入1ml冰冷的75％乙醇，离心10分钟，在4℃。 </li><li>完全弃去上清液并通过在离开管打开的盖干燥RNA沉淀。 不E:在这一点上的粒料不是清晰可见。仍要继续。 </li><li>重悬沉淀于20μl的分子级的水。通过测定使用的NanoDrop 260 nm和280nm处的吸收计算浓度和核酸纯度。 </li><li>通过使用任何传统的逆转录酶试剂盒，并按照manufacturer&#39;s协议认真着手合成cDNA。 注意:从4×10 4个幼稚的 CD4 + T细胞中获得约0.5-2微克的RNA。 cDNA可从100ng的RNA的被原料合成，并无需低RNA输入试剂盒在实时PCR反应中使用。 </li></ol>

<ol>
	<li>Mazzon, C., Viola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+tango+to+quadrilla:+current+views+of+the+immunological+synapse.">From tango to quadrilla: current views of the immunological synapse.</a> Cell Adh Migr. 1 (1), 7-12 (2007).</li><li>Hamann, D., Pa Baars, ., Hooibrink, B., van Lier, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+the+human+CD4++T-cell+population:+two+distinct+CD4++T-cell+subsets+characterized+by+coexpression+of+CD45RA+and+CD45RO+isoforms.">Heterogeneity of the human CD4+ T-cell population: two distinct CD4+ T-cell subsets characterized by coexpression of CD45RA and CD45RO isoforms.</a> Blood. 88 (9), 3513-3521 (1996).</li><li>Zhong, L., Zhang, Z., Lu, X., Liu, S., Chen, C. Y., Chen, Z. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NSOM+/+QD-Based+Visualization+of+GM1+Serving+as+Platforms+for+TCR+/+CD3+Mediated+T-Cell+Activation.">NSOM / QD-Based Visualization of GM1 Serving as Platforms for TCR / CD3 Mediated T-Cell Activation.</a> Biomed Res Int. , 276498 (2013).</li><li>Nagafuku, M., Okuyama, 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=PNAS+Plus:+CD4+and+CD8+T+cells+require+different+membrane+gangliosides+for+activation.">PNAS Plus: CD4 and CD8 T cells require different membrane gangliosides for activation.</a> Procs Natl Acad Sci USA. 109 (6), E336-E342 (2012).</li><li>Schnaar, R. L., Gerardy-Schahn, R., Hildebrandt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sialic+acids+in+the+brain:+gangliosides+and+polysialic+acid+in+nervous+system+development,+stability,+disease,+and+regeneration.">Sialic acids in the brain: gangliosides and polysialic acid in nervous system development, stability, disease, and regeneration.</a> Physiol Rev. 94, 461-518 (2014).</li><li>Villanueva-Cabello, T. M., Mollicone, R., Cruz-Mu&#241;oz, M. E., L&#242;pez-Guerrero, D. V., Mart&#236;nez-Duncker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+human+na&#239;ve+Th+cells+increases+surface+expression+of+GD3+and+induces+neoexpression+of+GD2+that+colocalize+with+TCR+clusters.">Activation of human na&#239;ve Th cells increases surface expression of GD3 and induces neoexpression of GD2 that colocalize with TCR clusters.</a> Glycobiology. 25 (12), 1454-1464 (2015).</li><li>M&#252;thing, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLC+in+structure+and+recognition+studies+of+glycosphingolipids.">TLC in structure and recognition studies of glycosphingolipids.</a> Methods Mol Biol. 76 (17), 183-195 (1998).</li><li>de Almeida, M. C., Silva, A. C., Barral, A., Barral Netto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+human+peripheral+blood+monocyte+isolation.">A simple method for human peripheral blood monocyte isolation.</a> Mem Inst Oswaldo Cruz. 95 (2), 221-223 (2000).</li><li>O'Neil-Andersen, N. J., Lawrence, 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=Differential+modulation+of+surface+and+intracellular+protein+expression+by+T+cells+after+stimulation+in+the+presence+of+monensin+or+brefeldin+A.">Differential modulation of surface and intracellular protein expression by T cells after stimulation in the presence of monensin or brefeldin A.</a> Clin Diagn Lab Immunol. 9 (2), 243-250 (2002).</li><li>Mannering, S. I., Zhong, J., Cheers, C. <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+activation,+proliferation+and+apoptosis+in+primary+Listeria+monocytogenes+infection.">T-cell activation, proliferation and apoptosis in primary Listeria monocytogenes infection.</a> Immunology. 106 (1), 87-95 (2002).</li><li>Marconi, S., Acler, M., 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=Anti-GD2-like+IgM+autoreactivity+in+multiple+sclerosis+patients.">Anti-GD2-like IgM autoreactivity in multiple sclerosis patients.</a> Mult Scler. 12 (3), 302-308 (2006).</li><li>Park, J. E., Wu, D. Y., 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=Fine+specificity+of+natural+killer+T+cells+against+GD3+ganglioside+and+identification+of+GM3+as+an+inhibitory+natural+killer+T-cell+ligand.">Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T-cell ligand.</a> Immunology. 123 (1), 145-155 (2008).</li><li>Simon, B. M., Malisan, F., Testi, R., Nicotera, P., Leist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disialoganglioside+GD3+is+released+by+microglia+and+induces+oligodendrocyte+apoptosis.">Disialoganglioside GD3 is released by microglia and induces oligodendrocyte apoptosis.</a> Cell Death Differ. 9 (7), 758-767 (2002).</li><li>Beske, O., Reichelt, M., Taylor, M. P., Kirkegaard, K., Andino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poliovirus+infection+blocks+ERGIC-to-Golgi+trafficking+and+induces+microtubule-dependent+disruption+of+the+Golgi+complex.">Poliovirus infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of the Golgi complex.</a> J Cell Sci. 120 (18), 3207-3218 (2007).</li><li>Zuber, C., Spiro, M. J., Guhl, B., Spiro, R. G., Roth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Golgi+apparatus+immunolocalization+of+endomannosidase+suggests+post-endoplasmic+reticulum+glucose+trimming:+implications+for+quality+control.">Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control.</a> Mol Biol Cell. 11 (12), 4227-4240 (2000).</li><li>Yamashiro, S., Okada, M., 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=Expression+of+(GD3+synthase)+gene+in+human+cancer+cell+lines:+high+level+expression+in+melanomas+and+up-regulation+in+activated+T+lymphocytes.">Expression of (GD3 synthase) gene in human cancer cell lines: high level expression in melanomas and up-regulation in activated T lymphocytes.</a> Glycoconj J. 12 (6), 894-900 (1995).</li><li>Wipfler, D., Srinivasan, G. V., 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=Differentially+regulated+expression+of+9-O-acetyl+GD3+(CD60b)+and+7-O-acetyl-GD3+(CD60c)+during+differentiation+and+maturation+of+human+T+and+B+lymphocytes.">Differentially regulated expression of 9-O-acetyl GD3 (CD60b) and 7-O-acetyl-GD3 (CD60c) during differentiation and maturation of human T and B lymphocytes.</a> Glycobiology. 21 (9), 1161-1172 (2011).</li><li>Pukel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., Old, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GD3,+a+prominent+ganglioside+of+human+melanoma.+Detection+and+characterisation+by+mouse+monoclonal+antibody.">GD3, a prominent ganglioside of human melanoma. Detection and characterisation by mouse monoclonal antibody.</a> J Exp Med. 155 (4), 1133-1147 (1982).</li></ol>

The authors have nothing to disclose.

所述协议可以用于本地化神经节苷脂或蛋白质中的 CD4 + T细胞或其它免疫细胞（ 例如 ，PMBCs， 图5）由少数细胞起始的细胞悬浮液。因为T细胞和非粘附性的小尺寸的，收购的信息差或低质量的荧光显微图像的结果，如果该细胞不正确地附着。 该协议具有良好质量的共聚焦显微镜分析相结合是对使用技术如薄层色谱法检测神经节苷脂的重要?...

的CD4 + T细胞协调通过其效应子功能由抗原呈递细胞1活化后的免疫应答。被激活期间调制的细胞机制的研究允许洞察免疫功能的一基本过程。然而，因为它们在血液周2代表细胞的非常小的群体幼稚CD4 + T细胞的研究可能是复杂的。 通过荧光显微镜几个报告已经研究参与CD4 + T细胞活化的不同分子，主要关联至质膜3蛋白的定位。神经节苷脂含唾液酸鞘糖脂，虽然他们已经在他们丰富，其他细胞等免疫细胞的神经细胞被广泛研究，也表达了与生物学相关职能4,5神经节苷脂。我们以前报道THA人幼稚CD4 + T细胞的活化有所述α2,8唾液酸转移酶ST8Sia 1（GD3合酶）和GM2 / GD2合酶的上调在t，诱导GD2的显著表面neoexpression和GD3神经节苷脂6的上调。在免疫细胞GD3，GD2和其他神经节苷脂的进一步研究是必要的，以补充免疫功能的蛋白质为基础的局部视图。 通常，神经节苷脂表达的研究是基于技术如薄层色谱法（TLC）7，但该技术不允许神经节苷脂的空间定位在质膜或在亚细胞区室，限制生物分析。 在这项工作中，我们描述了在人幼稚CD4 + T细胞和PBMC中抗CD3 /抗CD28活化后GD3和GD2神经节苷脂的抗体介导的识别和定位的协议。有了这个协议，我就也有可能以分析神经节苷脂的基因和分子表达在低数量在悬浮细胞，以获得高质量的图像6，考虑淋巴细胞的小尺寸（9微米）。

<table><tbody><tr><td>Advanced RPMI 1640&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>12633-012</td><td>Suplemented with 3% FBS</td></tr><tr><td>mouse anti human CD3 antibody</td><td>eBioscience</td><td>16-0037-81</td><td>OKT3 clone</td></tr><tr><td>mouse anti human CD28 antibody</td><td>ebioscience</td><td>16-0288-81</td><td>CD28.6 clone</td></tr><tr><td>mouse anti human GD3&amp;nbsp; antibody</td><td>Abcam</td><td>ab11779</td><td>R24 clone</td></tr><tr><td>mouse anti human GD2 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-53831</td><td>14G2a clone</td></tr><tr><td>FITC-conjugated anti-mouse IgG3&lt;br/&gt; antibody</td><td>Abcam</td><td>ab97259</td><td></td></tr><tr><td>Alexa Fluor 488 conjugated antimouse</td><td>Thermo Fisher Scieni&amp;iexcl;tific</td><td>A-21131</td><td></td></tr><tr><td>IgG2a antibody</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 647 conjugated antimouse</td><td>Thermo Fisher Scieni&amp;iexcl;tific</td><td>A-21241</td><td></td></tr><tr><td>IgG2a antibody</td><td></td><td></td><td></td></tr><tr><td>Hoechst 333258</td><td>Sigma-Aldrich</td><td>861405</td><td></td></tr><tr><td>&amp;nbsp;Ficoll Paque-Plus</td><td>GE</td><td>17-1440-02&amp;nbsp;</td><td></td></tr><tr><td>Trizol Reagent</td><td>Invitrogen</td><td>15596-026</td><td></td></tr><tr><td>Naive CD4+ T Cell Isolation Kit II, human</td><td>MACS Miltenyi Biotec</td><td>130-094-131</td><td></td></tr><tr><td>BD FACSAria II</td><td>BD Biosciences</td><td></td><td>Sorting&amp;nbsp;</td></tr><tr><td>Nunc Lab-tek chamber slide system</td><td>Sigma-Aldrich</td><td>C7182</td><td></td></tr><tr><td>Olympus FV 1000 Laser Confocal Microscope (Olympus)&amp;nbsp;</td><td>Olympus</td><td></td><td>Upright BX61WI and IX81&amp;nbsp;</td></tr><tr><td>Forward sequence primer GD3 synthase</td><td>5&amp;acute;-GAGCGTTCAGGAAACAAATGG- 3&amp;acute;</td><td></td><td>Ref. 7</td></tr><tr><td>Reverse sequence primer GD3 synthase</td><td>5&amp;acute;-CCTGTGGGAAGAGAGAGTAAG-3&amp;acute;</td><td></td><td>Ref. 7</td></tr><tr><td>Forward sequence primer GM2/GD2 synthase</td><td>5&amp;acute;-CAACACAGCAGACACAGTCC-3&amp;acute;</td><td></td><td>Ref. 7</td></tr><tr><td>Reverse sequence primer GM2/GD2 synthase</td><td>5&amp;acute;-GTGGCAATCGTGACTAGAGC-3&amp;acute;</td><td></td><td>Ref. 7</td></tr></tbody></table>

preparation of cd4+ t cells for analysis of gd3 and gd2 ganglioside membrane expression by microscopy

在悬浮液中和对幼稚CD4 + T细胞的活化本文描述的方法及其在盖玻片共聚焦显微镜分析依从允许参与CD4 + T细胞活化神经节苷脂的空间定位和可视化，即补体表达分析实验，如流式细胞仪，西印迹或实时PCR。神经节苷脂表达通过流式细胞仪，并通过显微镜它们的细胞定位的量化可以通过使用抗神经节苷脂抗体以高亲和力和特异性来获得。然而，在悬浮细胞的适当处理涉及培养板的处理，以促进对荧光或共聚焦显微镜获得所需的必要的粘附性。在这项工作中，我们描述了幼稚的 CD4 + T细胞活化期间确定GD3和GD2神经节苷脂表达并与TCR共定位的协议。此外，真正的-T使用&lt;40,000个细胞IME PCR实验为GD3和GM2 / GD2合酶基因的测定中描述，这表明基因分析实验可以低数量的细胞，无需额外的低输入的RNA试剂盒来进行。

在这个手稿中描述的协议使得培养良好的质量和附着静息和活化的人幼稚CD4 + T细胞（ 图1）。活化的CD4 + T细胞显示出相比于静止状态（ 图1A）的特征的增殖曲线（ 图1B）。的CD25迟活化标志是评价在通过共聚焦显微镜（ 图1C）观察到72小时的有效激活是有用的。的CD69标记当前通过流式细胞仪或显微镜用作?...

Watch this Scientific Journal Video about Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy at JoVE.com

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

我们描述了在显微镜使用，以确定在静止神经节苷脂和激活的人幼稚CD4 + T细胞的细胞膜表达和定位一个标准抗体染色方案。还使用&lt;40,000个细胞，不需要额外的低输入RNA试剂描述的实时PCR实验。

CD4的制备<sup&gt; +</sup&gt; T细胞通过显微镜分析GD3和GD2神经节苷脂膜表达的

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

preparation-cd4-t-cells-for-analysis-gd3-gd2-ganglioside-membrane

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

8.5K 次观看.  Universidad Autónoma del Estado de Morelos. 我们描述了在显微镜使用，以确定在静止神经节苷脂和激活的人幼稚CD4 + T细胞的细胞膜表达和定位一个标准抗体染色方案。还使用

视频: Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

可视化抗原特异性CD4 + T细胞的MHC II类四聚四

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

科研

生物学

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

原位检测自身反应性CD4 + T细胞在大脑和心脏使用主要组织相容性复合II类Dextramers的

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

免疫与感染

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

神经节苷脂提取、纯化和分析

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

生物化学

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

一<em&gt;在体外</em&gt;模型细胞病理生理学的球细胞脑​​白质营养不良的研究

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

神经科学

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

原生动物寄生虫感染原发性星细胞和微胶质的伴随分离

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

制备珠状支持脂质双分子层研究T细胞免疫突触的颗粒输出

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

A Fluorescence-Based Assay Using Engineered Lymphocytes for Screening Immunomodulatory Compounds

Source: Fouda, A., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55199">A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules.</a> J. Vis. Exp., (2017).
This video demonstrates the screening of immunomodulatory compounds via fluorescent-based assay. Transgenic mouse-derived T lymphocytes with green fluorescent protein expression cassettes are treated with these compounds. The enhanced and reduced GFP signals enable the identification of the compounds&#39; stimulatory and inhibitory effects, respectively.

使用工程淋巴细胞筛选免疫调节化合物的基于荧光的测定

1. Preparation of Splenocyte Medium and Flow-cytometry Buffer

	Perform all steps under a 70% ethanol-cleaned biological hood.
	Remove 70 mL of pre-warmed ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

An In Vitro Technique to Differentiate GM-CSF Producing T Helper Cells from Na&iuml;ve T Cells

Source: Lu, Y., et al.&#160;<a href="https://appjove.unicartagenaproxy.elogim.com/v/58087">In Vitro&#160;Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells.</a>&#160;J. Vis. Exp. (2018).
This video demonstrates the in vitro differentiation of granulocyte-macrophage-colony-stimulating factor or GM-CSF-producing T helper cells (THGM). Isolated na&#239;ve T cells are stimulated for differentiation into THGM, which is confirmed by detecting high intracellular GM-CSF production and a low expression of other T helper cell-specific cytokines.

一种体外技术，用于区分产生 GM-CSF 的 T 辅助细胞和幼稚 T 细胞

Source: Lu, Y., et al.&#160;In Vitro&#160;Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) ...

CD4的制备

摘要

探索更多视频

此视频中的章节