从牛皮癣患者的皮肤活检好心由Andreas Koerber博士经过科学使用口头或书面的知情同意书（在德国埃森大学皮肤科）提供。 XH也由国家自然科学基金委员会61263039和国家自然科学基金委员会11101321的支持。

注:从发生后科学运用口头或书面的知情同意择期整形手术个人腹部皮肤剩获得来自健康人的皮肤活检。利用人体皮肤的批准，并按照医学伦理委员会的Radboud大学医疗中心，奈梅亨，荷兰和德国埃森大学人类研究机构的规定。 1.准备从人体皮肤的单细胞悬浮液（工作在无菌流柜如果后续细胞培养是必需的） <ol><li>制备细胞培养基:RPMI 1640 +青霉素/链霉素（最终浓度100单位/ ml和100μg/ ml时，分别为）+丙酮酸（0.02毫摩尔）和Glutamax的（0.02毫摩尔），无血清加入。 </li><li>制备完全培养基:在步骤1.1±10％人合并的血清（HPS）中制备培养基;存放于4℃。摆在我们面前把中到20℃±2ING。 </li><li>使用4mm的圆形活检穿孔仪器获得的皮肤活检，并在20℃±2保持在RPMI 1640完全培养基长达4小时或在4℃下为ON。在实验室抵达后尽快处理活检。 注:较长皮肤的存储将影响细胞产量和细胞活力。 </li><li>标签的蓝色皑皑分离管加入5 ml完全培养基到的标记管。 </li><li>加2ml完全培养基的每孔（共3个孔）的无菌的6孔培养板的。用消毒工具活检放入一个单井，冲洗，将其移动到第二个好，重复此步骤，一个更多的时间，从而实现一共有三个冲洗。 </li><li>转移阱漂洗皮肤活检到无菌培养皿中，活检的顶部添加100μl的完全培养基中，并小心地刮去使用不锈钢一次性无菌解剖刀皮下脂肪组织中。 注:钍是是关键的一步。 </li><li>切割每个皮肤活检插入无菌培养皿4小块。转移样品（最多每管4毫米活组织检查四）含有5ml完全培养基的制备离解管。 </li><li>盖紧管的帽子，倒挂连接到自动组织离解的袖子。确保所有的样品材料位于转子的面积。 </li><li>通过执行"程序m_spleen _01"（由仪器的内部存储器或由伴有程序卡提供一个预先定义的节目）以​​解离活检在适当的旋转速度为56秒开始的解离过程。 </li><li>处理后，从离解器分离的解离管，确保所有的解离的材料在管的底部收集。 </li><li>添加150微升胶原酶IA（80毫克/毫升）到解离管孵育样品在振荡水浴中于37°下60分钟。添加抗酶的100微升（5 MU / ml）倒入thedissociation管，拌匀。 注:胶原酶或更长的温育时间将改变细胞生存力的浓度较高。 </li><li>附加离解管到自动组织离解的套筒和运行" 程序M_脾_01"解离 ​​活检一次。 </li><li>放置一个70微米尼龙细胞过滤器上的50ml Falcon管中的顶部。应用分离的样品材料，这个细胞滤网去除细胞团块/组织碎片。 </li><li>用5ml完全培养基洗细胞过滤一次。 C±2离心机在20℃，450×g离心10分钟，吸出上清液。 </li><li>重复该洗涤步骤一次。悬浮细胞沉淀在300微升完全培养基。单细胞悬液是准备用于进一步数据;继续与体外细胞培养协议或流式细胞分析。 </li><li>在进一步的情况下，我ntracellular细胞因子染色，刺激细胞用PMA（12.5毫微克/毫升）中，离子霉素（500纳克/毫升）和布雷非德菌素A（5微克/毫升）4小时，在37℃，5％CO 2培养箱用于执行流之前4小时仪分析。 </li></ol> 2.皮肤驻留性T细胞的活化多克隆（ 离体细胞培养） <ol><li>等份加入100μl单细胞悬液（在步骤1.15中制备）成圆底96孔板中。 注:对于每个4毫米皮肤活检，所获取的单细胞悬液可分成一个96孔板的至少两个孔中。 </li><li>添加抗CD3 /抗CD28单克隆抗体包被的微珠（每孔25000珠），重组人细胞因子的rIL-2（最终浓度25U / ml）和重组IL-1β（最终浓度为50ng / ml）中。 </li><li>覆盖良好的标记板盖培养板，孵育在5％CO 2，100％湿度，37 °C培养箱。当介质的颜色变成黄色更改网上平台。 <br/&gt;注:透明细胞集落可以从细胞培养的第8天进行观察。 </li></ol> 3.主要的流式细胞仪分析/培养的皮肤驻留性T细胞<ol><li>准备FACS缓冲液:PBS + 0.2％BSA。 </li><li>转移的目标细胞成V形底96孔板中。离心机在20℃±2，450×g离心2分钟，并吸出上清液。 </li><li>重悬在100μlPBS中的沉淀。离心机在20℃±2，450×g离心2分钟，并吸出上清液。 </li><li>染色的细胞用100μl缀合可定影活力染料制备eFluorescence780的:在4℃，30分钟（1 1000稀释使用PBS）中。添加100μl的FACS缓冲液，离心机在20℃±2，450×g离心2分钟，并吸出上清液。 </li><li>选择所需的细胞表面标记的单克隆抗体，例如:CD45-BV421（HI30，1:20），CD3-ECD（UCHT1，1:50），CD4-PC5.5（1388.2，1:200），CD8-APC-AlexFluo700 （B9.11，1:400），CD14-FITC（TUK4，1:50），CD19-APC-AlexFluo750（13-1191:50），CD56-PE（MEM-188，1:50），CD25-PeCy7（BC96，1:50），CD11c的-PeCy7（BU15，1:10），以及CD1C-APC（AD5-8E7， 1:10），根据测试的每种mAb稀释因子使用FACS缓冲液制备的单克隆抗体混合物。定义使用抗体的同型对照与非染色样品一起大门设置。 </li><li>添加25微升制备的单克隆抗体混合物的进每个孔中。孵育20分钟，在20℃±2，避光。 </li><li>添加100μl的FACS缓冲液，离心机在20℃±2，450×g离心2分钟，并吸出上清液。 </li><li>如需样品只需要细胞表面染色，继续执行步骤3.16。 </li><li>在细胞内的Foxp3染色的情况下: <ol><li>通过用3份的稀释剂的混合浓缩物中制备的固定和透化缓冲液。 </li><li>准备1个通透缓冲区:部分10X 1通透缓冲区+ 9份无菌H 2 O.的</li></ol></li><li>在100微升固定和通透性的悬浮颗粒zation缓冲，拌匀，并在4孵育 °下进行30分钟。 </li><li> 100μl的透化缓冲液添加至每个孔，离心在RT 450 xg离心2分钟，并吸出上清液。 </li><li>洗细胞用透化缓冲液一次。 </li><li>选择所需的细胞内的mAb，例如，Foxp3的-eFluo450（PCH1​​01，1:50），IL-17A-AlexFluo488（eBio64DEC1，1:50）和IFNγ-PeCy7（4S.B3，1:400）。使用根据测试每种mAb稀释因子含有2％正常大鼠血清透化缓冲液制备的单克隆抗体混合物。定义使用抗体的同型对照与非染色样品一起大门设置。 </li><li>加入20μl制备的单克隆抗体混合物的进每个孔中。孵育4 °℃，30分钟，避光。 </li><li>孵育30分钟后，加入100微升透化缓冲液到每个孔。离心机在20℃±2，450×g离心2分钟，并吸出上清液。 </li><li>洗净用透buffe细胞ř一次。重悬沉淀在110微升FACS缓冲液中，并转移细胞悬液到microFACS管。 注:样品是准备用10色流式细胞仪测量。 </li><li>在需要精确的细胞数的情况下，立即用流式细胞仪进行测量前加10微升fluorospheres的充分混合均匀的悬浮液到每个样本。 </li></ol>

<ol>
	<li>Clark, R. 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=The+vast+majority+of+CLA++T+cells+are+resident+in+normal+skin.">The vast majority of CLA+ T cells are resident in normal skin.</a> J Immunol. 176, 4431-4439 (2006).</li><li>Zaba, L. C., Fuentes-Duculan, J., Steinman, R. M., Krueger, J. G., Lowes, 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=Normal+human+dermis+contains+distinct+populations+of+CD11c+BDCA-1++dendritic+cells+and+CD163+FXIIIA++macrophages.">Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages.</a> J Clin Invest. 117, 2517-2525 (2007).</li><li>Gebhardt, T., 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=Memory+T+cells+in+nonlymphoid+tissue+that+provide+enhanced+local+immunity+during+infection+with+herpes+simplex+virus.">Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus.</a> Nature Immunol. 10, 524-530 (2009).</li><li>Boyman, O., 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=Spontaneous+development+of+psoriasis+in+a+new+animal+model+shows+an+essential+role+for+resident+T+cells+and+tumor+necrosis+factor-alpha.">Spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-alpha.</a> J Exp Med. 199, 731-736 (2004).</li><li>Christophers, E., Mrowietz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+infiltrate+in+psoriasis.">The inflammatory infiltrate in psoriasis.</a> Clin Dermatol. 13, 131-135 (1995).</li><li>Jiang, X., 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=Skin+infection+generates+non-migratory+memory+CD8++T(RM)+cells+providing+global+skin+immunity.">Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity.</a> Nature. 483, 227-231 (2012).</li><li>Havran, W. L., 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=Limited+diversity+of+T-cell+receptor+gamma-chain+expression+of+murine+Thy-1++dendritic+epidermal+cells+revealed+by+V+gamma+3-specific+monoclonal+antibody.">Limited diversity of T-cell receptor gamma-chain expression of murine Thy-1+ dendritic epidermal cells revealed by V gamma 3-specific monoclonal antibody.</a> Proc Natl Acad Sci U S A. 86, 4185-4189 (1989).</li><li>Campbell, J. 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The authors have nothing to disclose.

在这里，我们提出了一个协议，以有效地隔离人体皮肤活检的皮肤驻地T细胞。这个协议的优点是相对高的数字可行淋巴细胞的隔离，并表示相关的表面标记。确定的细胞亚群为:的CD11c +树突，CD4 +和CD8 + T细胞和Foxp3 + CD25 +细胞。重要的是，孤立的皮肤驻地T细胞的体外培养是很好的可行性，并允许进行后续功能分析。 人体皮肤可?...

皮肤，因为身体和环境之间的主要接口，提供了针对外部的物理，化学和生物损伤如伤人，紫外线辐射和微生物防御的第一道防线。皮肤由两个主要隔间，表皮和真皮层，并含有多种免疫细胞包括Langerhans细胞，巨噬细胞，树突细胞（DC），以及约20十亿记忆T细胞，本近两倍的数量在整个血容量1 ，2。数据越来越多支持皮肤有重要的免疫功能，无论是在组织的动态平衡和各种病理状态的概念。驻留在正常皮肤的免疫细胞被认为以进行免疫监视3，并已显示出在炎性疾病如牛皮癣4的发展中的作用。在银屑病皮损部位，CD4 +和CD8 +浸润T细胞OBServed并且它表明CD4和CD8的比例的变化取决于疾病状态5。但是，细胞的这些群体是很难研究，因为现有技术仅允许少数细胞的分离。 从人体皮肤T细胞分离当前广泛使用的技术相结合的机械皮肤解离酶处理。人体皮肤活检被广泛切碎，并与像胰蛋白酶，胶原酶和/或EDTA 6-8酶的培养。考虑到皮肤的阻挡薄纸这对拉力和机械解聚高度抗T细胞分离的确立方法产生非常少的细胞，并且甚至活细胞数目降低，这使得这些细胞群的体外细胞培养困难和具有挑战性的。 在这里，我们报告的改进方法结合mechan隔离来自健康和参与的银屑病人的皮肤淋巴细胞使用自动组织离解，而不是广泛的切碎建立的方法，一起用胶原酶酶消化皮肤的iCal分离。制备单细胞悬浮液后观察到的各种可行的免疫细胞亚群，包括DC和T细胞。重要的是，表面标记CD3，CD4和CD8的表达保存完好。由此制备的细胞，是准备用于体外细胞培养物或流式细胞分析使用。此协议已被成功地用于从牛皮癣患者的皮损衍生的单一皮肤活检（直径4 mm）的分析。结果表明，皮肤驻患者的T细胞产生更多的炎性细胞因子如IL-17和IFNγ在比较健康志愿者9。

<table border="0" cellpadding="0" cellspacing="0" width="843">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Name of Reagent/ Equipment</td>
			<td style="width:145px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:299px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch</td>
			<td>Kai Europe</td>
			<td>BP-40F</td>
			<td>4 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable sterile scalpels</td>
			<td>Dalhausen</td>
			<td>1100000510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS C tube</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
			<td>Blue-capped, used as the dissociation tube.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:281px;">gentleMACS Dissociator</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
			<td style="width:299px;">Automated tissue dissociator. Using the &#34;program spleen_01&#34; for dissociation of the skin biopsy.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer&#160;</td>
			<td style="width:145px;">BD</td>
			<td style="width:119px;">352350</td>
			<td>70 &#181;m nylon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">96-well U-bottom plate</td>
			<td style="width:145px;">Greiner Bio-One</td>
			<td style="width:119px;">650180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Life Technologies</td>
			<td>22409-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">GlutaMAX</td>
			<td>Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:281px;">Human Pooled Serum (HPS)</td>
			<td style="width:145px;">in house prepared</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Collagenase I</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">C2674</td>
			<td>Type 1-A, suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">DNase I</td>
			<td style="width:145px;">Calbiochem</td>
			<td style="width:119px;">260913</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PBS</td>
			<td style="width:145px;">B Braun</td>
			<td style="width:119px;">3623140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">BSA</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">A4503-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fixable Viability Dye (FVD) APC-eFluo780</td>
			<td>eBioscience</td>
			<td>65-0865-18</td>
			<td style="width:299px;">Stain dead cells prior to cell fixation; dilute with PBS at 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation/Permeabilization Concentrate</td>
			<td>eBioscience</td>
			<td>00-5123-43</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation/Permeabilization Diluent</td>
			<td>eBioscience</td>
			<td>00-5223-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permeabilization Buffer (10x)</td>
			<td>eBioscience</td>
			<td>00-8333-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">BV421 Mouse anti-human CD45</td>
			<td style="width:145px;">BD</td>
			<td style="width:119px;">563879</td>
			<td>Clone: HI30; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">FITC Mouse anti-human CD14</td>
			<td>Dako</td>
			<td style="width:119px;">T0844</td>
			<td>Clone: TUK4; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PE Mouse anti-human CD56</td>
			<td style="width:145px;">Dako</td>
			<td style="width:119px;">R7127</td>
			<td>Clone: MOC-1; dilution factor 1:50</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:281px;">ECD Mouse anti-human CD3</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A07748</td>
			<td style="width:299px;">Clone: UCHT1; dilution factor 1:50 for surface staining; dilution factor 1:25 for intracellular staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PC5.5 Mouse anti-human CD4</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">B16491</td>
			<td>Clone: 13B8.2; dilution factor 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PeCy7 Mouse anti-human CD11c</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A80249</td>
			<td>Clone: BU15; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">APC Mouse anti-human CD1c</td>
			<td style="width:145px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-090-903</td>
			<td>Clone: AD5-8E7; dilution factor 1:10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">APC-AlexFluo700 Mouse anti-human CD8</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A66332</td>
			<td>Clone: B9.11; dilution factor 1:400</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:281px;">APC-AlexFluo750 Mouse anti-human CD19</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A94681</td>
			<td>Clone: J3-119; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PeCy7 Mouse anti-human CD25</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">AD5-8E7</td>
			<td>Clone: BC96; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">&#160;PE Rat anti-human CLA</td>
			<td style="width:145px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-091-635</td>
			<td>clone: HECA-452; dilution factor 1:</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">eFluo450 Rat anti-human Foxp3</td>
			<td>eBIoscience</td>
			<td>48-4776-42</td>
			<td style="width:299px;">Clone: PCH101; intracellular staining; dilution factor 1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">AlexFluo488 Mouse anti-human IL-17A</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">53-7179-42</td>
			<td style="width:299px;">Clone: eBio64DEC17; intracellular staining; dilution factor 1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">PeCy7 Mouse anti-human IFNg</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">25-7319-41</td>
			<td style="width:299px;">Clone: 4S.B3; intracellular staining; dilution factor 1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Flow-Count Fluorospheres</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td align="right" style="width:119px;">7547053</td>
			<td>Counting beads, for flow cytometry</td>
		</tr>
	</tbody>
</table>

lymphocyte isolation from human skin for phenotypic analysis and ex vivo cell culture

人体皮肤具有重要的屏障功能，并包含有助于从病原体组织稳态和保护各种免疫细胞。由于皮肤相对容易获得，它提供了一个理想的平台，研究外周免疫调节机制。免疫固有细胞在健康的皮肤进行免疫监视，而且还起到炎性皮肤疾病，如牛皮癣的发展中起重要作用。尽管新兴的见解，我们对生物学基本各种炎症皮肤病的了解仍然有限。有必要从皮肤活检样本中分离出质量好（单）的细胞群。到目前为止，隔离程序已经严重缺乏获得足够数量的活菌的阻碍。隔离和随后的分析也已受到的免疫细胞谱系标志物的损失，由于引起的电流离解过程来获得机械和化学应力单细胞悬浮液。在这里，我们描述了一种改进方法，通过使用一种自动组织离解器和胶原酶处理组合机械皮肤分离以分离从健康的和涉及银屑病人类皮肤T细胞。这种方法在单细胞悬浮液的制备保持最免疫谱系标记，例如CD4，CD8，Foxp3的和CD11c的表达。成功的CD4 + T细胞的分离和随后的表型和功能的分析的例子被示出。

这里介绍的协议将产生2200±178000 760之间的615（平均值±SEM，健康志愿者的皮肤）至±用一个4 mm皮肤活检时（平均值±SEM，牛皮癣患者的皮损）从人体皮肤可行的淋巴细胞。 不同类型的 CD45 +细胞在来自健康个体包括CD4 + T细胞（〜45％），CD8 + T细胞（〜30％）的皮肤衍生的单细胞悬液进行鉴定，和的CD11c +树突状（〜5％ ），而少数B细胞（...

Watch this Scientific Journal Video about Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture at JoVE.com

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

lymphocyte-isolation-from-human-skin-for-phenotypic-analysis-ex-vivo

Research

JoVE Journal

Immunology and Infection

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

14.4K 次观看.  Radboud University Medical Centre. We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

视频: Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA - IG基于人工抗原提呈细胞可高效体外扩展人类的CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

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

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

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

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

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 ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

单细胞悬液的细胞荧光分析从不同的鼠标皮肤区域准备

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

分离和<em&gt;离体</emVδ1的&gt;企业文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT细胞，胸腺外的αβT细胞祖

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

小鼠淋巴细胞通过标签<sup&gt; 64</sup&gt;的Cu-抗体受体靶向用于<em&gt;体内</em&gt;由PET / CT细胞运输

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

外迁移系统中淋巴细胞迁移的评估

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

从表型分析和人体皮肤淋巴细胞分离

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迁移人T淋巴细胞分离，培养和分析