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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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?...

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 border="0" cellpadding="0" cellspacing="0" width="1213">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:457px;">HBSS with phenol red, without calcium, without magnesium, liquid</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">55021C</td>
			<td style="width:396px;">Keep sterile until day of usage.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">HEPES, &#8805;99.5% (titration)</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">H3375</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:457px;">EDTA disodium salt solution</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">E7889</td>
			<td style="width:396px;">Keep sterile until day of usage.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:457px;">Fetal Bovine Serum, USDA, Heat Inactivated, Premium Select</td>
			<td style="width:179px;">MidSci</td>
			<td style="width:181px;">S01520HI</td>
			<td style="width:396px;">Keep sterile until day of usage.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:457px;">Percoll, pH 8.5-9.5 (25&#176;C)</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">P1644</td>
			<td style="width:396px;">Keep sterile. Percoll needs to be made isotonic with sterile 10X PBS prior to use.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:457px;">Trimmer Combo Kit</td>
			<td style="width:179px;">Kent Scientific</td>
			<td style="width:181px;">CL9990-1201&#160;</td>
			<td style="width:396px;">Use the larger trimmer for shaving the flank and back.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:457px;">4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">E0753-10G</td>
			<td style="width:396px;">Dissolve in 100% EtOH at 50&#176;C for 15 minutes on a rotating plate.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:457px;">Purified anti-mouse CD16/CD32 (2.4G2)</td>
			<td style="width:179px;">Tonbo Biosciences</td>
			<td style="width:181px;">70-0161</td>
			<td style="width:396px;">Antibody used to block non-specific antibody binding during antibody staining for flow cytometry</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:457px;">anti-CD3e-BV650 (145-2C11)</td>
			<td style="width:179px;">Becton Dickinson</td>
			<td style="width:181px;">564378</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD4-APC-eFluor780 (RM4-5)</td>
			<td style="width:179px;">eBioscience</td>
			<td style="width:181px;">47-0042-80</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD8a-BV785 (53-6.7)</td>
			<td style="width:179px;">BioLegend</td>
			<td style="width:181px;">100749</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD11b-eFluor450 (M1/70)</td>
			<td style="width:179px;">eBioscience</td>
			<td style="width:181px;">48-0112-80</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD11c-eFluor450 (N418)</td>
			<td style="width:179px;">eBioscience</td>
			<td style="width:181px;">48-0114-80</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD44-BV711 (IM7)</td>
			<td style="width:179px;">Becton Dickinson</td>
			<td style="width:181px;">563971</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD45-FITC (30-F11)</td>
			<td style="width:179px;">Tonbo Biosciences</td>
			<td style="width:181px;">35-0451-U025</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD45R-eFluor450 (RA3-6B2)</td>
			<td style="width:179px;">eBioscience</td>
			<td style="width:181px;">48-0452-80</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD62L-PerCP-Cy5.5 (MEL-14)</td>
			<td style="width:179px;">BioLegend</td>
			<td style="width:181px;">104431</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-Ly-6G/Ly-6C (Gr-1)-PE (RB6-8C5)</td>
			<td style="width:179px;">BioLegend</td>
			<td style="width:181px;">108407</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">Ghost Dye-BV510</td>
			<td style="width:179px;">Tonbo Biosciences</td>
			<td style="width:181px;">13-0870-T100</td>
			<td style="width:396px;">Viability dye for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">LSR Fortessa</td>
			<td style="width:179px;">Becton Dickinson</td>
			<td style="width:181px;">N/A</td>
			<td style="width:396px;">Cell analyzer (18 parameters)</td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:457px;">Collagenase D from Clostridium histolyticum</td>
			<td style="width:179px;">Roche Applied Science</td>
			<td style="width:181px;">11088858001</td>
			<td style="width:396px;">Aliquot lyophilized enzyme at 5 mg/mL in HBSS with phenol red, without calcium, and without magnesium into 1 mL aliquots.&#160; Store immediately at -20&#176;C for up to six months.</td>
		</tr>
		<tr height="92">
			<td height="92" style="height:92px;width:457px;">ND4 Swiss female mice</td>
			<td style="width:179px;">Harlan</td>
			<td style="width:181px;">0-32</td>
			<td style="width:396px;">8-12 weeks old; conventionally housed with free access to food and water and used according to Macalester College&#39;s IACUC guidelines.&#160;</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（?...

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

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 and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

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

Isolation 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 Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脑幽静的白细胞的分离与分析<em&gt;伯氏疟原虫</emANKA感染的小鼠

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

生物能量和氧化突发:协议用于人类白细胞和血小板的隔离与评价

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

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 ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

从人类母胎界面分离白细胞

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...