Name: isolation of myeloid dendritic cells and epithelial cells from human thymus
Uploaded: 2013-09-19T21:00:00
Description: Watch this Scientific Journal Video about Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus at JoVE.com

我们感谢胸心血管外科系，大学医院图宾根的外科医生提供的CDR2抗体为我们提供了胸腺样品和布鲁诺Kyewski（DKFZ，海德堡，德国）。我们还要感谢汉斯 - 约尔格Bühring和萨布丽娜格林从分拣设施（图宾根大学）。这项工作是由SFB 685和Hertie基金会的支持。

1。工具，酶溶液和缓冲液的制备执行开始的协议之前，可用以下制备步骤。 <ol><li>工具 清洁，干燥，高压灭菌下列工具，让他们在无菌包装备用。 <ol><li>小尖剪刀切割的胸腺组织或者弯曲或直的提示。小弯钳与处理组织锯齿状的提示。 </li><li> 50毫升橡树岭离心管，PC，对珀密度离心步骤。 </li></ol></li><li>酶溶液 准备胶原酶/ DNA酶I酶混合物（2毫克胶原酶/ ml和0.1毫克?...

<ol>
	<li>Miller, J. F. A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+discovery+of+thymus+function+and+of+thymus-derived+lymphocytes.">The discovery of thymus function and of thymus-derived lymphocytes.</a> Immunological Reviews. 185, 7-14 (2002).</li><li>Klein, L., Hinterberger, M., Wirnsberger, G., Kyewski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+presentation+in+the+thymus+for+positive+selection+and+central+tolerance+induction.">Antigen presentation in the thymus for positive selection and central tolerance induction.</a> Nat Rev Immunol. 9, 833-844 (2009).</li><li>Vandenabeele, S., Hochrein, H., Mavaddat, N., Winkel, K., Shortman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+thymus+contains+2+distinct+dendritic+cell+populations.">Human thymus contains 2 distinct dendritic cell populations.</a> Blood. 97, 1733-1741 (2001).</li><li>Gotter, J., Brors, B., Hergenhahn, M., Kyewski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medullary+Epithelial+Cells+of+the+Human+Thymus+Express+a+Highly+Diverse+Selection+of+Tissue-specific+Genes+Colocalized+in+Chromosomal+Clusters.">Medullary Epithelial Cells of the Human Thymus Express a Highly Diverse Selection of Tissue-specific Genes Colocalized in Chromosomal Clusters.</a> The Journal of Experimental Medicine. 199, 155-166 (2004).</li><li>Rouse, R. V., Bolin, L. M., Bender, J. R., Kyewski, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+reactive+with+subsets+of+mouse+and+human+thymic+epithelial+cells.">Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells.</a> Journal of Histochemistry &amp; Cytochemistry. 36, 1511-1517 (1988).</li><li>Stoeckle, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cathepsin+S+dominates+autoantigen+processing+in+human+thymic+dendritic+cells.">Cathepsin S dominates autoantigen processing in human thymic dendritic cells.</a> Journal of Autoimmunity. 38, 332-343 (2012).</li><li>Woods Ignatoski, K. M., Bingham, E. L., Frome, L. K., Doherty, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+trans-differentiation+of+thymus+cells+into+parathyroid-like+cells+without+genetic+manipulation.">Directed trans-differentiation of thymus cells into parathyroid-like cells without genetic manipulation.</a> Tissue Eng Part C Methods. 17, 1051-1059 (2011).</li><li>Williams, K. 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=Single+Cell+Analysis+of+Complex+Thymus+Stromal+Cell+Populations:+Rapid+Thymic+Epithelia+Preparation+Characterizes+Radiation+Injury.">Single Cell Analysis of Complex Thymus Stromal Cell Populations: Rapid Thymic Epithelia Preparation Characterizes Radiation Injury.</a> Clinical and Translational Science. 2, 279-285 (2009).</li><li>Bendriss-Vermare, N., 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=Human+thymus+contains+IFN-&#945;-producing+CD11c-,+myeloid+CD11c+,+and+mature+interdigitating+dendritic+cells.">Human thymus contains IFN-&#945;-producing CD11c-, myeloid CD11c+, and mature interdigitating dendritic cells.</a> The Journal of Clinical Investigation. 107, 835-844 (2001).</li><li>Schmitt, N., 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=Ex+vivo+characterization+of+human+thymic+dendritic+cell+subsets.">Ex vivo characterization of human thymic dendritic cell subsets.</a> Immunobiology. 212, 167-177 (2007).</li><li>Dzionek, A., 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=BDCA-4:+Three+Markers+for+Distinct+Subsets+of+Dendritic+Cells+in+Human+Peripheral+Blood.">BDCA-4: Three Markers for Distinct Subsets of Dendritic Cells in Human Peripheral Blood.</a> The Journal of Immunology. 165, 6037-6046 (2000).</li><li>Wu, L., Shortman, K. <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+thymic+dendritic+cells.">Heterogeneity of thymic dendritic cells.</a> Seminars in Immunology. 17, 304-312 (2005).</li><li>Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+enzymes+improve+isolation+and+characterization+of+the+adult+thymic+epithelium.">Purified enzymes improve isolation and characterization of the adult thymic epithelium.</a> Journal of Immunological Methods. 385, 23-34 (2012).</li><li>Adamopoulou, E., Tenzer, S., Hillen, N., Klug, P., Rota, I. A., Tietz, S., Gebhardt, M., Stevanovic, S., Schild, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+MHC-peptide+matrix+of+central+tolerance+in+the+human+thymus.">Exploring the MHC-peptide matrix of central tolerance in the human thymus.</a> Nature Communications. , (2013).</li></ol>

这里所描述的协议是协议由Gotter 等 4公布了修改。在协议中的关键步骤是条件和组织的初始准备以及Percoll密度分离。我们强烈建议尽快收集处理后的组织中。在清洗和切割组织时，工作速度快，但是彻底是很重要的。在步骤2.3中所述的胸腺细胞洗涤，至关重要的是将压力施加与对组织片注射器柱塞的背面过于剧烈和长时间的压力会损坏基质细胞时，以找到合适的平衡。分层介质上?...

胸腺是在T细胞发育发生器官。随着年龄的增长其相对和绝对尺寸减小，当它变得先后被脂肪取代，虽然胸腺活性仍然可以在旧时代被检测到。其对免疫反应的重要性表现在20世纪60年代初1。 对T细胞库是通过T细胞受体与不同种类的胸腺的APC，其提供生存或死亡的线索来发展T细胞，导致功能性，在很大程度上自耐受T细胞库2肽-MHC复合物的相互作用形成的。 约98％的细胞在人类胸腺正在开发称为胸腺T细胞。剩余的2％由许多不同的细胞类型，包括各种TEC的（皮质，延髓，囊下），粒细胞和浆细胞样DC（MDC，PDC）的巨噬细胞，B细胞，成熟重新循环T细胞，粒细胞， fibroblasts，内皮细胞和用表达的表型非常罕见的上皮细胞类似，从其他组织如肌肉，神经和呼吸上皮（ 图1）的细胞。这些中，TEC和DC处于正常胸腺中发现的主要的APC类型。近年来，这些APC类型的文化和分子分析的净化已经获得了越来越多的关注。由于其低频率中的任进行详细的分析，这些细胞类型的分离需要高效的，可重现的和成本有效的方法。这里介绍的方法是从先前公布的研究3,4的修改。 与任何其他组织，从胸腺细胞提取可以通过酶促解聚的细胞 - 细胞和细胞 - 基质相互作用的网络，以便获得单细胞悬浮液来实现。有一定的参数，如良好的分离效率，细胞产量，细胞活力和保持细胞的S需要urface标记物是至关重要的，并为这些稀有细胞群的成功分离进行优化。 在这个协议中，直流和TEC亚群分离是通过使由机械破碎和酶消化组织的单细胞悬浮液进行。我们用胶原酶溶组织梭菌 ，其中有不同的酶活性比例的平衡，打破了保持组织在一起的天然胶原。 DNA酶I包括在酶溶液，以减少细胞聚集由于从死细胞的游离DNA（胸腺是非常敏感的），我们还提供了另一种方法，在典型的酶消化组织，涉及机械和酶组织治疗由组织离解的协助。单细胞悬浮液，然后进行单Percoll密度离心细胞的低密度部分（LDF）的富集。从细胞中的该级分，DC可以通过染色F是孤立或DC表面标记物（ 即表面CD11c +）和使用磁性分离或荧光激活细胞分选（FACS）。不像含有细胞在胸腺中的绝大多数的淋巴样细胞，TEC不以高水平表达CD45，但正对上皮细胞粘附分子EpCAM的。 CTEC可以从髓质TEC通过对CDR-2（皮质树突网状细胞-2）抗体4,5和稍低的EpCAM表达识别的尚未定义的抗原的表达来区分。差动共表达的EpCAM和CDR2的允许这些TEC的子集通过高速细胞的高效分离排序6。 这里介绍的协议被优化用于人类胸腺组织。该过程的持续时间取决于组织的量和实验者的能力以及细胞分选器的速度，如果FACS分选被使用。通常情况下，该协议的直流隔离可以在5完成-6小时和TEC在8-10小时的隔离。直流和TEC亚群的胸腺组织中分离对时间很敏感。更快的分离过程中，细胞的状况就越好。最后，可以用于进一步调查喜欢的mRNA和比较研究蛋白的表达，PCR实验，蛋白分离，分子图谱（ 即转录，微RNA分析），以及细胞培养物6的分离的细胞。 操守准则为了能够与人体胸腺组织工作，研究人员需要自组织通常是由未成年人获得了当地伦理委员会或主管部门以及捐助者的知情同意书（或通常是他或她的父母，获得批准孩子）。此外，所有人体组织应作为应采取潜在的传染和适当的措施，如戴手套工作， 等处理。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and Materials</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>PAA</td>
			<td>E15-842</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>PAA</td>
			<td>H15-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum-Gold</td>
			<td>PAA</td>
			<td>A15-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>PAA</td>
			<td>K41-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10 103 586 001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I, grade II bovine pancreatic</td>
			<td>Roche</td>
			<td>10 104 159 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 10x in PBS</td>
			<td>PAA</td>
			<td>L11-001</td>
			<td>stock conc. 20 mg/ml</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Protein Labelling kit</td>
			<td>Molecular Probes</td>
			<td>A-10235</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human CDR2 (purified)</td>
			<td>Bruno Kyewski, DKFZ- Heidelberg, Germany</td>
			<td></td>
			<td>labeled with Alexa Fluor 488</td>
		</tr>
		<tr>
			<td>anti-human CD45 (Pacific Blue)</td>
			<td>Biolegend</td>
			<td>304022</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human EpCAM (APC)</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-254</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human CD11c (PE)</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-411</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-PE Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45 Microbeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-045-801</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td>for tissue dissociator</td>
		</tr>
		<tr>
			<td>Percoll (density 1.130 g/ml)</td>
			<td>GE Healthcare, Life Sciences</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile distilled Water (DNAse/ RNAse free)</td>
			<td>GIBCO</td>
			<td>10977-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamunex 10%</td>
			<td>Tajecris-Biotherapeutics</td>
			<td>G120052</td>
			<td>1:10 pre-dilution, use 20 &#956;l/1 x 106cells</td>
		</tr>
		<tr>
			<td>0.22 &#956;m filter</td>
			<td>Millex GS</td>
			<td>SLGS033SS</td>
			<td>Syringe driven</td>
		</tr>
		<tr>
			<td>Stericup filter unit</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td>Pump driven</td>
		</tr>
		<tr>
			<td>50 ml PC oak ridge centrifuge tubes</td>
			<td>Nalgene</td>
			<td>3118-0050</td>
			<td>50 ml</td>
		</tr>
		<tr>
			<td>50 ml PP conical tubes</td>
			<td>Becton Dickinson</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm x 75 mm 5 ml test tubes</td>
			<td>Becton Dickinson</td>
			<td>352058</td>
			<td>FACS stainings</td>
		</tr>
		<tr>
			<td>Cell strainer 70 &#956;m</td>
			<td>Becton Dickinson</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>INSTRUMENTS</td>
		</tr>
		<tr>
			<td>Flow Cytometer-Sorter (BD FACSAriaTMIIu)</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Evolution R6 (rotor)</td>
			<td>Kendro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator REAX 2</td>
			<td>Heidolph</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-093 235</td>
			<td>tissue dissociator</td>
		</tr>
	</tbody>
</table>

isolation of myeloid dendritic cells and epithelial cells from human thymus

在这个协议中，我们提供了一个方法，从人胸腺分离的树突状细胞（DC）和上皮细胞（TEC）。 DC和TEC是主要的抗原呈递细胞在正常的胸腺发现（APC）的类型，它是公认的，他们胸腺选择过程中扮演不同的角色。这些细胞被定位在不同的微环境中的胸腺和各APC型拼成细胞中，只有轻微的人口。为了进一步了解这些类型的细胞生物学，这些细胞群的特征是非常可取的，但由于它们的低频率，任何这些类型的细胞分离需要一个高效的和可再现的过程。这个协议的细节的方法，以获得适合不同的细胞特性表征细胞。胸腺组织被机械破坏和之后各个步骤的酶消化，将所得细胞悬浮液用Percoll密度离心步骤富集。对于髓样DC（CD11c的隔离&lt;SUP&gt; +），从低密度部分（LDF）细胞通过磁性细胞分选免疫选择。 TEC人群（MTEC，CTEC）的富集是通过从低密度的Percoll细胞部分，允许其通过荧光激活细胞分选（FACS），使用特定细胞标志物随后分离耗尽造血（CD45 hi）条的细胞来实现。将分离的细胞可以用于不同的下游应用。

如本协议的原料，我们使用胸腺组织从知情同意后，并根据机构准则得到孩子接受矫正心血管外科（胸心血管外科杂志，大学诊所图宾根处）除去。这个废弃的材料可以大大的大小从2-30 g以上而有所不同。得到了的mDC的数量和TEC的子集（CTEC和MTEC）取决于大小，以及用于隔离的胸腺组织样品的寿命。 图2显示了（〜9cm）中从一个六岁的孩子获得了相当大的一块组?...

Watch this Scientific Journal Video about Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus at JoVE.com

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

这个协议的细节的方法来分离抗原通过不同的步骤的组织随后在单细胞悬浮液的密度离心和感兴趣的细胞群的最后磁和/或FACS分选的酶消化从人胸腺呈递细胞。

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Research

JoVE Journal

Immunology and Infection

Isolation of Mouse Lung Dendritic Cells

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

科研

免疫与感染

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

性T细胞Transinfection从树突状细胞捕捉细菌

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征：两个单核细胞分离方法的比较

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â±

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

高盐抗原表达树突状细胞的分离和采用转移

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

从人类唾液腺中分离唾液表皮细胞,作为脂质层或单层体进行体外生长

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...