Name: lymphocyte isolation from human skin for phenotypic analysis and ex vivo cell culture
Uploaded: 2016-04-08T13:00:00.000+00:00
Duration: 10 min 31 s
Description: Watch this Scientific Journal Video about Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture at JoVE.com

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. J., 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=CCR7+expression+and+memory+T+cell+diversity+in+humans.">CCR7 expression and memory T cell diversity in humans.</a> J Immunol. 166, 877-884 (2001).</li><li>He, 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=Targeting+PKC+in+human+T+cells+using+sotrastaurin+(AEB071)+preserves+regulatory+T+cells+and+prevents+IL-17+production.">Targeting PKC in human T cells using sotrastaurin (AEB071) preserves regulatory T cells and prevents IL-17 production.</a> J Invest Dermatol. 134, 975-983 (2014).</li><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=A+novel+method+for+the+isolation+of+skin+resident+T+cells+from+normal+and+diseased+human+skin.">A novel method for the isolation of skin resident T cells from normal and diseased human skin.</a> J Invest Dermatol. 126, 1059-1070 (2006).</li><li>Clark, R. A., Kupper, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+and+dermal+fibroblasts+induce+proliferation+of+natural+regulatory+T+cells+isolated+from+human+skin.">IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin.</a> Blood. 109, 194-202 (2007).</li><li>Keijsers, R. R., 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=In+vivo+induction+of+cutaneous+inflammation+results+in+the+accumulation+of+extracellular+trap-forming+neutrophils+expressing+RORgammat+and+IL-17.">In vivo induction of cutaneous inflammation results in the accumulation of extracellular trap-forming neutrophils expressing RORgammat and IL-17.</a> J Invest Dermatol. 134, 1276-1284 (2014).</li><li>Hybbinette, S., Bostrom, M., Lindberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+dissociation+of+keratinocytes+from+human+skin+biopsies+for+in+vitro+cell+propagation.">Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation.</a> Experimental dermatology. 8, 30-38 (1999).</li><li>Hennino, 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=Skin-infiltrating+CD8++T+cells+initiate+atopic+dermatitis+lesions.">Skin-infiltrating CD8+ T cells initiate atopic dermatitis lesions.</a> J Immunol. 178, 5571-5577 (2007).</li></ol>

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><tbody><tr><td>Disposable Biopsy Punch</td><td>Kai Europe</td><td>BP-40F</td><td>4 mm</td></tr><tr><td>Disposable sterile scalpels</td><td>Dalhausen</td><td>1100000510</td><td></td></tr><tr><td>gentleMACS C tube</td><td>Miltenyi Biotech</td><td>130-093-237</td><td>Blue-capped, used as the dissociation tube.</td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotech</td><td>130-093-235</td><td>Automated tissue dissociator. Using the &quot;program spleen_01&quot; for dissociation of the skin biopsy.</td></tr><tr><td>Cell strainer&amp;nbsp;</td><td>BD</td><td>352350</td><td>70 &amp;micro;m nylon</td></tr><tr><td>96-well U-bottom plate</td><td>Greiner Bio-One</td><td>650180</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>22409-015</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Life Technologies</td><td>11360-039</td><td></td></tr><tr><td>GlutaMAX</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Human Pooled Serum (HPS)</td><td>in house prepared</td><td></td><td></td></tr><tr><td>Collagenase I</td><td>Sigma-Aldrich</td><td>C2674</td><td>Type 1-A, suitable for cell culture</td></tr><tr><td>DNase I</td><td>Calbiochem</td><td>260913</td><td></td></tr><tr><td>PBS</td><td>B Braun</td><td>3623140</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A4503-500G</td><td></td></tr><tr><td>Fixable Viability Dye (FVD) APC-eFluo780</td><td>eBioscience</td><td>65-0865-18</td><td>Stain dead cells prior to cell fixation; dilute with PBS at 1:1,000</td></tr><tr><td>Fixation/Permeabilization Concentrate</td><td>eBioscience</td><td>00-5123-43</td><td></td></tr><tr><td>Fixation/Permeabilization Diluent</td><td>eBioscience</td><td>00-5223-56</td><td></td></tr><tr><td>Permeabilization Buffer (10x)</td><td>eBioscience</td><td>00-8333-56</td><td></td></tr><tr><td>BV421 Mouse anti-human CD45</td><td>BD</td><td>563879</td><td>Clone: HI30; dilution factor 1:50</td></tr><tr><td>FITC Mouse anti-human CD14</td><td>Dako</td><td>T0844</td><td>Clone: TUK4; dilution factor 1:50</td></tr><tr><td>PE Mouse anti-human CD56</td><td>Dako</td><td>R7127</td><td>Clone: MOC-1; dilution factor 1:50</td></tr><tr><td>ECD Mouse anti-human CD3</td><td>Beckman - Coulter</td><td>A07748</td><td>Clone: UCHT1; dilution factor 1:50 for surface staining; dilution factor 1:25 for intracellular staining.</td></tr><tr><td>PC5.5 Mouse anti-human CD4</td><td>Beckman - Coulter</td><td>B16491</td><td>Clone: 13B8.2; dilution factor 1:200</td></tr><tr><td>PeCy7 Mouse anti-human CD11c</td><td>Beckman - Coulter</td><td>A80249</td><td>Clone: BU15; dilution factor 1:50</td></tr><tr><td>APC Mouse anti-human CD1c</td><td>Miltenyi Biotech</td><td>130-090-903</td><td>Clone: AD5-8E7; dilution factor 1:10</td></tr><tr><td>APC-AlexFluo700 Mouse anti-human CD8</td><td>Beckman - Coulter</td><td>A66332</td><td>Clone: B9.11; dilution factor 1:400</td></tr><tr><td>APC-AlexFluo750 Mouse anti-human CD19</td><td>Beckman - Coulter</td><td>A94681</td><td>Clone: J3-119; dilution factor 1:50</td></tr><tr><td>PeCy7 Mouse anti-human CD25</td><td>eBioscience</td><td>AD5-8E7</td><td>Clone: BC96; dilution factor 1:50</td></tr><tr><td>&amp;nbsp;PE Rat anti-human CLA</td><td>Miltenyi Biotech</td><td>130-091-635</td><td>clone: HECA-452; dilution factor 1:</td></tr><tr><td>eFluo450 Rat anti-human Foxp3</td><td>eBIoscience</td><td>48-4776-42</td><td>Clone: PCH101; intracellular staining; dilution factor 1:50</td></tr><tr><td>AlexFluo488 Mouse anti-human IL-17A</td><td>eBioscience</td><td>53-7179-42</td><td>Clone: eBio64DEC17; intracellular staining; dilution factor 1:50</td></tr><tr><td>PeCy7 Mouse anti-human IFNg</td><td>eBioscience</td><td>25-7319-41</td><td>Clone: 4S.B3; intracellular staining; dilution factor 1:400</td></tr><tr><td>Flow-Count Fluorospheres</td><td>Beckman - Coulter</td><td>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

14.5K 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

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

荧光活化细胞分选分离人皮肤状和网状成纤维细胞

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

遗传学

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

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 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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活<em&gt;疟原虫</em&gt;孢子

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

医学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

生物学

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

从小鼠真皮淋巴毛细血管中分离内皮细胞

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

作者聚焦：基于磁珠的小鼠真皮淋巴内皮细胞分离

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

小鼠真皮淋巴内皮细胞分离

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

从表型分析和人体皮肤淋巴细胞分离

摘要

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迁移人T淋巴细胞分离，培养和分析

荧光活化细胞分选分离人皮肤状和网状成纤维细胞

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

单细胞悬液的细胞荧光分析从不同的鼠标皮肤区域准备

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活

人淋巴管内皮细胞中的多参数荧光激活细胞分选分离

成人从人体皮肤的上皮干细胞的分离和培养

小鼠小肠免疫系统淋巴细胞的分离

小鼠真皮淋巴内皮细胞分离

从表型分析和人体皮肤淋巴细胞分离

摘要

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此视频中的章节

迁移人T淋巴细胞分离，培养和分析

荧光活化细胞分选分离人皮肤状和网状成纤维细胞

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

单细胞悬液的细胞荧光分析从不同的鼠标皮肤区域准备

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

成人从人体皮肤的上皮干细胞的分离和培养

小鼠小肠免疫系统淋巴细胞的分离

小鼠真皮淋巴内皮细胞分离

人淋巴管内皮细胞中的多参数荧光激活细胞分选分离