美国国立卫生研究院（NIH R15 NS067536-01A1直流），国家慢性阴部疼痛协会（奖DC），以及麦卡莱斯特学院支持这项工作。 CB获得了奖学金从麦卡利斯特大学贝克曼学者计划，由阿诺德和梅布尔贝克曼基金会资助。 BTF和TM是由JDRF 2-2011-662支持。我们感谢贾森申克尔博士和朱丽安娜·刘易斯博士的技术咨询，以及Chatterjea实验室的所有现任和前任成员的帮助和支持。

注:8-12周龄雌性ND4 Swiss Webster小鼠，传统安置与免费获得食物和水，用于这些研究中使用（B13S1）实验方案经麦卡莱斯特学院的IACUC。 1.致敏和用恶唑酮<ol><li> 0天<ol><li>通过加入3ml异氟烷来放置网格下方在一个4升带盖玻璃瓶的底部吸收巾制备麻醉室中。对于这里使用的ND4雌性小鼠，时间在腔室是30-60秒直到物体被适当麻醉;优化麻醉给药所使用的小鼠品系。 </li><li>剃须每个麻醉小鼠的背部和侧面使用动物毛发修剪。 </li></ol></li><li>第1天<ol><li>使4-乙氧基亚甲基-2-苯基-2-恶唑啉-5-酮（牛）成绝对乙醇的2％溶液，孵育在50℃下15分钟上的旋转板在培养箱中。 </li><li>简言之麻醉使用如1.1.1所述，并应用100微升2％，牛年要在各约20微升多个串行应用剃光回异氟烷麻醉小鼠。等待串行应用之间5-10秒，将溶液干燥。 </li><li>小心避免溢出上的区域比背面以外的2％牛溶液，并等待该溶液应用程序之间进行干燥。 </li></ol></li><li> 5-7天<ol><li>使（牛）的无水乙醇的1％溶液，孵育在50℃下15分钟上的旋转板在培养箱中。 </li><li>轻轻但稳固地固定所述小鼠在同腹颈部和尾部基颈背朝上和颈部稍稍倾斜向下（类似于一个保持为腹膜内注射）和应用100微升1％牛到剃侧翼在几个串行应用和抽空皮肤干燥之后，如上所述。 </li><li>对于对照小鼠，应用100微升无水乙醇车辆到剃坯ķ。 </li></ol></li></ol> 2.侧翼皮肤丰收<ol><li>安乐死使用二氧化碳气体对小鼠，并小心地切除皮肤侧面（〜25毫米×25毫米的皮肤）和10厘米手术剪的希望的区域。 </li><li>使用干净，锋利的手术刀片，刮去从侧翼皮肤多余的脂肪和结缔组织。 </li><li>放置3个侧面皮肤的来自3只小鼠到50毫升锥形管含有10ml的RT的HBSS [用酚红，不含钙或镁，5mM的乙二胺四乙酸（EDTA），10mM的4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES），和10％胎牛血清（FBS）。 </li></ol> 3.预消化组织清洗<ol><li>地方管含有皮肤片段上的旋转板30分钟在保持在37℃的干燥的非充气培养箱。 </li><li>涡大力10秒在温育期结束。 </li><li>通过一个70微米的应变的管中的内容过滤器丢弃洗涤缓冲液和收集组织。一个单一的过滤器可以在整个过程中重复使用生物处理组中。 </li><li>用一对镊子，传送从过滤侧翼皮肤进入一个新的50ml锥形管中含有10毫升新鲜，RT的HBSS培养基（如上所述含有EDTA，HEPES，和FBS）。 </li><li>用12.5厘米一对浸入管外科解剖剪刀的，剁碎每片侧翼皮肤使平均片组织是约2.2毫米×2.2毫米的区域。 </li><li>地方管含有皮肤片段上的旋转板30分钟在保持在37℃的干燥的非充气培养箱。 </li><li>涡大力10秒在孵育结束时</li></ol>皮肤4.胶原酶消化<ol><li>通过一个70微米的滤网过滤管的内容物，收集和侧翼皮片转移到一个新的50ml锥形管含有10ml FRE的sh，RT HBSS介质补充有0.7毫克/毫升胶原酶D。 </li><li>地方管含有在保持在37℃的干燥的非充气培养箱消化介质和皮肤片段上的旋转板30分钟。 </li><li>管剧烈10秒的在温育结束涡流内容。 </li><li>管通过一个70微米的过滤器过滤的内容到一个新的50ml锥形管;保持流通含有消化的组织并丢弃过滤器和侧翼皮肤碎屑。 </li><li> 5分钟，在350克在50毫升HBSS介质，离心机通过流洗，倾倒出上清液。 </li></ol> 5.密度梯度离心<ol><li>使用血清吸管，在1X磷酸盐缓冲盐水（PBS）加入3 ml的室温的67％的密度梯度离心介质为一个新的15毫升锥形管，用于每个样品。 </li><li>重悬细胞沉淀在5ml RT 44％的密度梯度离心介质在HBSS与酚红。 </li><li>根特LY层5毫升含有在67％的密度梯度离心介质样品的顶部的细胞用血清吸管的44％密度梯度离心介质。确保设置移液器最慢的速度，然后将吸管沿着锥形管的壁上。要小心，不要动摇，破坏或反转梯度。 </li><li>设置加速到最低设置，松开刹车，离心梯度在931 XG 20分钟，在室温确保离心机是平衡的。 </li><li>离心后，小心地从离心机取出梯度，并轻轻地运送到实验台上拿着管子直立。使用5毫升塑料移液管，在44％和67％的密度梯度离心介质和传输的界面除去细胞层，以一个新的15毫升锥形管中。如果接口是不可见的，只需在67％-44％的边界取出约2毫升的液体。 </li><li>填充锥形管从INTE含有细胞rface有2％FBS在1X冰冷的PBS，离心细胞5分钟，在350×g离心，倾倒出上清液。 注意:这是第一次发现的细胞可以被冷却/保冷自消化步骤是在37℃和密度梯度离心介质的步骤是在室温。细胞可以在一个1被再悬浮:1台盼蓝稀释和计数和活力的信息可以在此时获得。 </li></ol> 6.阻塞和染色的流式细胞仪分析<ol><li>块FcγRII/ III受体的细胞上，以防止非特异性抗体染色。对于这里描述的实验中，使抗体预混在PBS，2％FBS的含1:100稀释抗CD16 / 32（2.4G2）未结合的抗体结合和块FcγRII/Ⅲ受体。 </li><li>孵育阻断细胞的抗体对CD3ɛ（145-2C11），CD4（RM4-5），CD8α（53-6.7）的CD11b（M1 / 70），表面CD11c（N418），CD44（IM7），CD45（适当的预混30-F11），CD45R（RA3-6B2），CD62L（MEL-14），和LY-6G / LY-6C（GR-1）（RB6-8C5），以及亮紫510活/死染色，以确定死亡细胞。使用所有抗体无论是在推荐的制造商或以前的研究人员优化稀释。对于这里所描述的实验中，使用的所有抗体以1:100的稀释。 </li><li>在50μl的1X PBS染色缓冲液含2％FBS洗涤细胞，重悬。保持在冰上，直至荧光数据可以在流式细胞仪上获得。 注意:有补偿参数预先优化采集荧光数据的前一个淋巴组织和一个共同的细胞表面抗原（如CD4或CD8）是很重要的。 </li><li>以增加细胞数量为下游流式细胞分析，从染色的细胞的整个样品采集数据。 </li></ol> 7.使用标准方法来完成下面列出的步骤分析流式细胞仪数据<ol><li>首先，对前向散射的淋巴细胞区域栅极（区）通过侧散射（区）的情节。 </li><li>然后，选择使用前向散射（宽）由侧散射（宽）的单细胞，避免分析双峰。这也可以实现与由侧向散射高度情节前方散射高度。 </li><li>然后，在谱系栅极（细胞CD11b，CD11c的，和CD45R / B220）阴性和CD3阳性事件，要排除的巨噬细胞，树突细胞，和B细胞，分别和局限分析到T细胞区室，如果目标是，因为它是在这里，以评估浸润T细胞。 </li><li>接着，在活细胞排除活/死染色栅极。 </li><li>最后，在所感兴趣的细胞群栅极（见代表性结果部分）。 </li></ol>

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作者有没有财务或其他利益的披露。

表征变化，皮肤的居民白细胞皮肤疾病的啮齿动物模型，如特应性皮炎或银屑病是理解炎症细胞大量涌入和疾病病理之间的机械连接非常重要。在这里，我们描述了一个经济的技术来隔离皮肤组织的白细胞在大多数生物医学研究实验室发现基本设备。这种较快的技术避免了使用昂贵的组织分离机和自管和试剂，有助于节约资源，同时尽量减少手工操作时间在实验室工作台上。温和酶促消化和不连续梯度分离除去大部分上皮细胞和碎片，和分离的细胞可用于各种的下游应用，包括流式细胞仪分析，细胞分选，转移，细胞培养和体外刺激测定法。消化酶与胶原酶D有对T细胞表面标志完整，preser无影响VES细胞活力。这个协议可以很容易地修改，以处理从区域比侧翼其他皮肤。 在此过程中的最重要的步骤是:1）彻底除去任何脂肪和结缔组织而收获皮肤，2）的处理和加工组织迅速仔细最大化浸润细胞产量，同时最小化的细胞死亡，3）轻轻分层的梯度，和4）提取渐变的接口。在开始大规模的实验它练密度梯度离心介质分层和接口去除是重要的。一个可以练习从一个不连续密度梯度收获细胞，通过70微米过滤器进入的1X PBS染色缓冲液破碎整个鼠脾脏和按照步骤5如上所述进行密度梯度这是很容易地看到和提取之间的界面44％的密度梯度离心介质和使用藻67％的密度梯度离心介质lenocytes，由于大量存在的免疫细胞。 当使用密度梯度工作，气泡应避免同时移液，和梯度中断应最小。锥形管含有梯度应轻拿轻放，并在任何时候都保持垂直。密度梯度离心媒体解决方案应始终在室温，设置为最慢的释放速度，保持在室温的离心机，设置为低加速度脱开制动，并仔细血清学移液器纺前平衡。当皮肤制剂能产生白细胞浸润的少量工作，细胞并不总是在梯度界面中可见。因此，重要的是，使44％的密度梯度离心介质用HBSS含有酚红使得接口能够容易地可视化，甚至当层细胞不能被分辨。此外，要温柔，但解压时迅速，清洗是非常重要的和处理皮肤组织，以避免细胞死亡之前的皮肤被放置在HBSS介质。 如果细胞活力或纯度低，更短的（约20分钟）的消化时间，胶原酶D（〜0.5毫克/毫升），或更精细切碎的组织稍低浓度可能会有所帮助。由于过度消化和组织的退化可能导致细胞的死亡，调查人员将需要优化的最小消化时间，提供了一个足够大的细胞样本量17。这个过程可能会需要在个别研究者的手上多轮优化，标准化的技术。胶原酶D活性水平还可以制造大量的和调整，以消化时间之间变化将有每批酶用来制成。在优化过程中，重要的是要进行纯化，用和不用的酶消化步骤与可靠的活/死染色以及免疫细胞染色林参考细胞悬浮液（例如脾）EAGE标记（例如CD45，CD3ɛ，细胞CD11b，CD11c的，等）以确保细胞群和感兴趣表面标志物穿过组织消化过程被保留。如果可能的话，它是有帮助的上一个流执行优化仪，可以测量正向和侧向散射的宽度或高度，以及面积，以排除细胞聚集。最后，细胞悬浮液应在光学显微镜的视觉可行性评估和一般的细胞形态下进行检查。所述细胞可以手动使用光学显微镜下或在流式细胞仪上使用计数珠0.4％台盼蓝染料排除进行计数。对于需要特定细胞类型的更严格的定量，在切除的组织的研究中，研究人员可以权衡或之前和消化后测量组织碎片的体积，并用消化的程度，以更准确地估计本细胞亚群数量在组织中的存在进行分析。 这方面的一个限制协议是，它特别适用于免疫细胞的纯化。如果一个人的爱好分离，纯化，和另一细胞群体（例如，皮肤上皮细胞）的分析，密度梯度的设置和酶促消化协议都将有可能需要进行修改和优化，以最好的隔离感兴趣的细胞。然而，该协议允许隔离两个淋巴样和髓样细胞亚群和纯化，因而可以适应大多数免疫的应用程序。另一个限制是，此协议不能使用的细胞群渗入真皮与皮肤的表皮之间进行区分。为了实现这种区别水平，该协议将必须进一步适于与额外的步骤来酶促分离真皮和表皮的前或代替此处所述的步骤。这里，将样品从〜3只小鼠汇集，以促进多参数流式细胞仪分析使得难以检测生物复制之间的差异。然而，根据所选择的下游应用，该协议可以很容易地修饰以产生和使用的样品从单个受试者。最后，该协议在这里为年轻呈现，ND4雌性小鼠可能需要进行修改，针对不同年龄，性别或菌株根据研究人员的需要的小鼠。 综上所述，温和的酶的这样的组合消化和不连续梯度分离提供从炎性皮损小鼠纯化的免疫细胞的一个简单，有效和经济的方法。它可直接或穿过皮肤病症的啮齿动物模型的一个不同的范围宽泛地适于作为一种方便的工具，以评估免疫浸润并分离白细胞的纯的，可行的人口为下游应用。

皮肤状况，从接触性皮炎，湿疹，牛皮癣，蜂窝组织炎，真菌感染和脓肿，以非黑色素瘤皮肤癌被认为是世界范围内的50个最流行 ​​的疾病之一，非致命疾病于2010年1第四全球领先的原因。因此，底层的多样皮肤病理学分子和细胞机制的调查是研究的一个必要的和活跃的领域。啮齿动物模型已在炎性皮肤状况的了解非常有用的，例如特应性皮炎2，牛皮癣3，或金黄色葡萄球菌感染 4。对于小鼠皮肤组织的酶消化廉价，高效，且简单的协议可以提供细胞，它可以用于各种下游应用，以便更好地了解皮肤疾病的病理生理的制剂。这里，一个简单而经济的方法被描述为小鼠皮肤的酶消化组织和皮肤浸润白细胞的隔离，可以用来进行细胞培养， 在体内继转移，流式细胞仪分析和分选或基因表达研究。此过程的总的目标是制备皮肤浸润白细胞单细胞悬浮液与高细胞生存力，同时尽量减少通常与定制试剂盒和机械dissociators相关的成本。 现有的皮肤组织裂解方法5-7可能导致低细胞活力及表面标志的完整性，或需要定制酶试剂盒和昂贵的组织分离机8-11。而鼠耳皮肤组织的消化是合理流行12-13消化高度角化皮肤组织（例如，从侧面）可导致污染大量非细胞碎片的细胞制剂。在最近的一项研究中，扎伊德和同事在2.5毫克消化鼠标侧面的皮肤90分钟/ ml的分散酶，弗洛45分钟，在3毫克/毫升胶原酶7结婚。在另一项研究中，这些研究人员使用的多个孵育，用2.5小时的结合消化，包括使用胰蛋白酶/ EDTA，胶原酶III的和分散酶5。不推荐用于酶促消化皮肤使用胰蛋白酶，与来自不同制造商的胰蛋白酶治疗已经显示出可测量的影响对哺乳动物细胞14-15的细胞表面蛋白的完整性。此外，分散酶可以对CD4和CD8αT细胞增殖的能力显著作用和影响的至少20分子的表面丰度，包括常见的T细胞活化标记物如CD62L 16。其他协议使用RPMI 1640在消化介质 6。然而， 镁离子和钙离子的在RPMI的存在会引起广泛的细胞聚集17。 一个理想的协议，用于组织分离的目标应该是高细胞活力，低细胞聚集，和损害最小的水平与细胞表面蛋白。高质量淋巴结基质细胞的准备工作已经完成，从而使用较短的酶孵育，钙2+和Mg 2+免费媒体协议，并避免胰蛋白酶和分散酶18。然而，这种类型的协议尚未建立用于全鼠皮肤的解离。 这里，一个协议描述以解离，隔离和从过敏原挑战小鼠侧翼皮肤丰富皮肤浸润白细胞。简言之，将切下的皮肤是预孵育中Hank氏平衡盐溶液（HBSS）中，用10％胎牛血清1小时，以软化组织的消化和除去任何过量的死皮或脂肪组织。这之后是30分钟的酶消化步骤与0.7毫克/毫升胶原酶D.胶原酶D具有对细胞表面标记物密度的影响最小，并在体外 16,18对T细胞增殖没有影响，使得它非常适用于涉及表面蛋白的表征的应用程序。下列酶消化，不连续密度梯度离心法从单细胞悬浮液除去上皮细胞和碎片和富集造血细胞。重要的是，此过程避免了昂贵的基于列的磁性细胞分离试剂和组织离解机8-11，并且可以与设备和材料在碱性生物医学研究实验室发现来执行。此处这个协议是用于分离白细胞从侧翼皮肤挑战三次半抗原恶唑酮（牛）在先前致敏ND4 Swiss小鼠（改编自19）。使用多参数流式细胞仪分析细胞。这种技术产生了细胞悬浮液以最小的碎片和分离的淋巴细胞的&gt; 95％的存活率这是由多参数流式细胞术分析来测量的T淋巴细胞和嗜中性粒细胞渗入影响SK在。

<table><tbody><tr><td>HBSS with phenol red, without calcium, without magnesium, liquid</td><td>Sigma Aldrich</td><td>55021C</td><td>Keep sterile until day of usage.</td></tr><tr><td>HEPES, &amp;ge;99.5% (titration)</td><td>Sigma Aldrich</td><td>H3375</td><td></td></tr><tr><td>EDTA disodium salt solution</td><td>Sigma Aldrich</td><td>E7889</td><td>Keep sterile until day of usage.</td></tr><tr><td>Fetal Bovine Serum, USDA, Heat Inactivated, Premium Select</td><td>MidSci</td><td>S01520HI</td><td>Keep sterile until day of usage.</td></tr><tr><td>Percoll, pH 8.5-9.5 (25&amp;deg;C)</td><td>Sigma Aldrich</td><td>P1644</td><td>Keep sterile. Percoll needs to be made isotonic with sterile 10X PBS prior to use.</td></tr><tr><td>Trimmer Combo Kit</td><td>Kent Scientific</td><td>CL9990-1201&amp;nbsp;</td><td>Use the larger trimmer for shaving the flank and back.</td></tr><tr><td>4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one</td><td>Sigma Aldrich</td><td>E0753-10G</td><td>Dissolve in 100% EtOH at 50&amp;deg;C for 15 minutes on a rotating plate.</td></tr><tr><td>Purified anti-mouse CD16/CD32 (2.4G2)</td><td>Tonbo Biosciences</td><td>70-0161</td><td>Antibody used to block non-specific antibody binding during antibody staining for flow cytometry</td></tr><tr><td>anti-CD3&amp;epsilon;-BV650 (145-2C11)</td><td>Becton Dickinson</td><td>564378</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD4-APC-eFluor780 (RM4-5)</td><td>eBioscience</td><td>47-0042-80</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD8&amp;alpha;-BV785 (53-6.7)</td><td>BioLegend</td><td>100749</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD11b-eFluor450 (M1/70)</td><td>eBioscience</td><td>48-0112-80</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD11c-eFluor450 (N418)</td><td>eBioscience</td><td>48-0114-80</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD44-BV711 (IM7)</td><td>Becton Dickinson</td><td>563971</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD45-FITC (30-F11)</td><td>Tonbo Biosciences</td><td>35-0451-U025</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD45R-eFluor450 (RA3-6B2)</td><td>eBioscience</td><td>48-0452-80</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD62L-PerCP-Cy5.5 (MEL-14)</td><td>BioLegend</td><td>104431</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-Ly-6G/Ly-6C (Gr-1)-PE (RB6-8C5)</td><td>BioLegend</td><td>108407</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>Ghost Dye-BV510</td><td>Tonbo Biosciences</td><td>13-0870-T100</td><td>Viability dye for flow cytometry</td></tr><tr><td>LSR Fortessa</td><td>Becton Dickinson</td><td>N/A</td><td>Cell analyzer (18 parameters)</td></tr><tr><td>Collagenase D from &lt;I&gt;Clostridium histolyticum&lt;/I&gt;</td><td>Roche Applied Science</td><td>11088858001</td><td>Aliquot lyophilized enzyme at 5 mg/ml in HBSS with phenol red, without calcium, and without magnesium into 1 mL aliquots.&amp;nbsp; Store immediately at -20&amp;deg;C for up to six months.</td></tr><tr><td>ND4 Swiss female mice</td><td>Harlan</td><td>0-32</td><td>8-12 weeks old; conventionally housed with free access to food and water and used according to Macalester College&#039;s IACUC guidelines.&amp;nbsp;</td></tr></tbody></table>

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.

胶原酶D处理的脾细胞显示出的CD4和CD8 T细胞上的 α 类似的水平 相比，媒体处理的对照 首先，胶原酶D对的对T细胞亚群谱系和活化标记的频率和表面丰度的任何潜在影响使用次级淋巴组织作为对照进行了评估。从ND4小鼠得到洗涤1小时用HBSS介质脾细胞悬浮液。接着，一半的细胞进行0.7毫克/毫升胶原酶D（如上述第4节中所述）。剩余的细胞重新悬浮在HBSS中控制平台。接着，这两个脾细胞级分使用密度梯度离心如上述第5节中描述进行处理。然后将细胞用抗表面CD4和CD8α染色，并在流式细胞仪分析。单身，住，CD45 +，非T系- （B220 -细胞CD11b -的CD11c - ），CD3ɛ+ 细胞进行门控以进行分析，以排除B细胞，巨噬细胞，树突状细胞。细胞和30％的CD4 - - CD4和CD8α蛋白脾细胞的表面上对已进行胶原酶ð消化和那些尚未曝光用约60％ 的 CD4 +CD8α酶上同样检测到检测CD8α+细胞与任一处理（ 图1A）。因此，胶原酶ð消化并没有影响CD4和CD8α的相对频率。表面的CD4和CD8α几何平均荧光单位（GMFI）对这些子集的强度也比较的两种治疗之间; GMFI值的CD4分别为2271分别酶处理和2243为控制脾细胞，并GMFI值CD8α分别为536酶处理的和520进行控制的脾细胞。酶处理也对T细胞活化的表面密度没有影响标记物ÇD44或CD62L在CD4 + T细胞 （图1B）。为CD44几何平均荧光强度值分别为669酶处理和654控制脾细胞; GMFI值的CD62L是1523为酶处理和1517为控制脾细胞。作为胶原酶ð消化保留在分离的细胞表面蛋白的完整性，这里描述的方案可以用于对下游流式细胞分析信心。 酶消化和侧面的皮肤细胞产生高细胞活力梯度净化 上述获得的细胞悬浮液的细胞产量和百 ​​分比生存力用台盼蓝染料排除20评估。这种消化和梯度分离协议产生约250可行的每侧翼组织毫米2牛年攻击小鼠的免疫细胞，约4可行的每毫米组织的2车辆攻击小鼠的免疫细胞先前致敏牛（ 表1）。 酶消化和侧面的皮肤细胞产生强劲复苏的免疫细胞的梯度纯化 接着，在皮肤的免疫细胞浸润的程度通过分析细胞的牛挑战的小鼠和对照小鼠，以评估使用此处描述的方法皮肤浸润免疫细胞恢复的侧面分离的CD45表面蛋白的丰度进行测定。 CD45是一个泛造血谱系标记21。 95％的低前向和侧向散射，单细胞（选通未示出）在牛攻击小鼠，并且这些细胞在车辆挑战对照的71％为活CD45 +细胞（ 图2）。在侧翼皮肤以下使用本文描述的方法（表1）3每日牛挑战检测浸润CD45 +免疫细胞阿〜67倍的增加。 ="1"&gt;三侧翼牛挑战导致T细胞和嗜中性粒细胞的显着浸润 我们的技术产生了从侧面皮肤的细胞群，可用于检测多种骨髓和淋巴细胞亚群的诸如的Gr-1 +嗜中性粒细胞22，CD4 + T细胞和CD8αT细胞。我们观察到〜6倍，7〜倍，和〜73倍增加，中性粒细胞，CD4 + T细胞，和CD8αT细胞，分别以下3每日牛挑战（ 表1）。我们所获得的活细胞的总数乘以计算这些增加由给定的免疫细胞片段的百分比出存活细胞栅极的期间流式细胞分析（使用台盼蓝排除计数）。然后我们将活总免疫细胞在牛处理的小鼠的数量，的Gr-1 +，CD4 +，或CD8α+细胞由相应的数字为车辆的对照小鼠，使我们倍增加淋巴细胞亚群。的Gr-1 +嗜中性粒细胞占单，现场CD45 + B220 42％ -的CD11b - CD11c的-细胞在牛治疗的小鼠和该群体在EtOH处理的小鼠的10％ （图2B）。在牛处理的小鼠，〜门控CD3 + T细胞的52％是CD8α+和 〜34％为CD4 +，而在车辆的控制，〜T细胞的9％为CD8α+和 57％的患者的 CD4 +（图2C） 。因此，利用该技术此处描述的，过敏性和非过敏性皮肤侧翼可被处理，以产生适合于浸润免疫细胞亚群的高分辨率流式细胞表征单细胞悬浮液。 <table fo:keep-together.within-page="1"><tbody><tr><td> 从鼠标侧面的皮肤细胞亚群 </td><td> 牛/牛（3） </td><td> 牛/乙醇（3） </td><td> 折叠扩展（牛/ Ethanol） </td></tr><tr><td></td><td colspan="3"> 每mm 2组织 </td></tr><tr><td>细胞的总数量</td><td> 262.7 </td><td> 5.3 </td><td> 49.3 </td></tr><tr><td> CD45 +免疫细胞</td><td> 250.7 </td><td> 3.8 </td><td> 66.7 </td></tr><tr><td> CD4 + CD8 -细胞</td><td> 6.5 </td><td> 0.9 </td><td> 7.2 </td></tr><tr><td> CD4 - CD8 +细胞 </td><td> 10.6 </td><td> 0.1 </td><td> 72.9 </td></tr><tr><td>中性粒细胞</td><td> 8.6 </td><td> 1.5 </td><td> 5.6 </td></tr></tbody></table> 从表中ND4雌性小鼠过敏性皮肤的侧翼1.细胞的产量 。使用台盼蓝排除活细胞从侧面皮肤中分离进行计数，并且标准化为细胞基于皮肤分离的区和小鼠的数目/ mm 2的组织每那朵汇集起诉的准备。我们计算出的总的免疫细胞产量的基础上检测基于谱系特异性表面标志物的造血抗原CD45在细胞表面上，和淋巴细胞亚群的产量。然后我们把细胞在牛年治疗的小鼠的数量由车辆控制老鼠的数量，给我们折增加淋巴细胞亚群。示出的数据表示来自每个治疗2-3只小鼠汇集的数据，和代表两个独立的实验。 <img alt="图1" src="/files/ftp_upload/53638/53638fig1.jpg" /> 脾细胞的图1.酶消化和梯度纯化保存细胞亚群和细胞活化标记。脾细胞洗涤1小时用HBSS介质，孵育或者胶原酶D或单独的HBSS介质和使用密度梯度以除去碎片和红血细胞中纯化。 T细胞被确定为住，CD45 +，血统（B220，细胞CD11b，CD11c的）&lt;SUP&gt; - ，和CD3ɛ+。 CD4和CD8αT细胞的频率酶消化（A）的后没有改变。 CD44和CD62L在CD4 + T细胞表面密度并没有改变下列酶促消化（B）中。显示的数据表示从2-3只小鼠的治疗从单一的实验汇总数据。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53638/53638fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53638/53638fig2.jpg" /> 图2.酶消化和皮肤细胞的梯度净化揭示控制与过敏原的挑战鼠标侧面的皮肤明显的免疫细胞浸润。三翼牛挑战产生皮肤浸润的免疫细胞在以前致敏ND4瑞士小鼠（A〜67倍A）。三牛的挑战还提供图片oked中的Gr-1 +嗜中性粒细胞（B）的一个〜6倍的增加，和〜73-和〜7倍CD8α+和 CD4 + T细胞，分别（C）的增加。现场CD45 +免疫细胞从单一的，低正向和侧向散射单元门;中性粒细胞和T细胞B220门-细胞CD11b - CD11c的-活的单个细胞。 1台盼蓝稀释以下梯度密度分离:计数采用一个1执行。显示的数据表示，从2-3小鼠各治疗组汇总数据，并代表了两个独立的实验。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53638/53638fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation at JoVE.com

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

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

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.

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

isolation-infiltrating-leukocytes-from-mouse-skin-using-enzymatic

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

21.2K 次观看.  Macalester College. 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. 

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

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

脑浸润白细胞的分离

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

科研

免疫与感染

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

小鼠皮肤角质细胞的分离和染色,用于细胞周期特定分析细胞蛋白表达的量度细胞测定

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

发育生物学

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

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

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 ...

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 ...

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 ...

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 ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

使用胶原酶E从毛里结肠分离拉米纳普里亚单核细胞

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

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 ...

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

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