Name: preparation of cd4+ t cells for analysis of gd3 and gd2 ganglioside membrane expression by microscopy
Uploaded: 2016-11-08T13:00:00
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...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="1052">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">Advanced RPMI 1640&#160;</td>
			<td style="width:241px;">Thermo Fisher Scientific</td>
			<td style="width:137px;">12633-012</td>
			<td style="width:193px;">Suplemented with 3% FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human CD3 antibody</td>
			<td>eBioscience</td>
			<td>16-0037-81</td>
			<td>OKT3 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human CD28 antibody</td>
			<td>ebioscience</td>
			<td>16-0288-81</td>
			<td>CD28.6 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human GD3&#160; antibody</td>
			<td>Abcam</td>
			<td>ab11779</td>
			<td>R24 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human GD2 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53831</td>
			<td>14G2a clone</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:481px;">FITC-conjugated anti-mouse IgG3 
			antibody</td>
			<td>Abcam</td>
			<td>ab97259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 conjugated antimouse</td>
			<td>Thermo Fisher Scieni&#161;tific</td>
			<td>A-21131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2a antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647 conjugated antimouse</td>
			<td>Thermo Fisher Scieni&#161;tific</td>
			<td>A-21241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2a antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 333258</td>
			<td>Sigma-Aldrich</td>
			<td>861405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Ficoll Paque-Plus</td>
			<td>GE</td>
			<td>17-1440-02&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Naive CD4+ T Cell Isolation Kit II, human</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-094-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACSAria II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Sorting&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab-tek chamber slide system</td>
			<td>Sigma-Aldrich</td>
			<td>C7182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus FV 1000 Laser Confocal Microscope (Olympus)&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Upright BX61WI and IX81&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward sequence primer GD3 synthase</td>
			<td>5&#180;-GAGCGTTCAGGAAACAAATGG- 3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse sequence primer GD3 synthase</td>
			<td>5&#180;-CCTGTGGGAAGAGAGAGTAAG-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward sequence primer GM2/GD2 synthase</td>
			<td>5&#180;-CAACACAGCAGACACAGTCC-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse sequence primer GM2/GD2 synthase</td>
			<td>5&#180;-GTGGCAATCGTGACTAGAGC-3&#180;</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 ...

Research

JoVE Journal

Immunology and Infection

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

科研

免疫与感染

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of NaÃƒÆ’Ã‚Â¯ve CD4 T Lymphocytes

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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

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

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 ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...