NKS is supported by NIH award K08 AI108690.

所有研究均按照机构动物护理和使用委员会（IACUC）在哈佛医学院，符合由美国协会为实验动物科学（AALAS）设定的标准，兽药受到严格的审查和指导原则进行的。 1.通过合房细菌的水平传输（可选） <ol><li>为了尽量减少外源性污染（特别是如果采用悉生只），实行无菌技术操作，而组装一次性无菌笼子，用食物和都被蒸压水。 </li><li>使用耳钻或标签单独标记6周龄C57BL / 6小鼠窝藏不同microbiotas，并在同一个笼子里容纳他们。由于老鼠是食粪，吃从笼底粪便颗粒，微生物自然会水平的小鼠之间传输。 </li><li>联合家鼠为≥1一周，以便有时间分析前微生物转移和免疫学变化。 </li></OL&gt; 从小肠上皮内层和固有层单细胞悬液的2.准备工作<ol><li> 溶液的制备 <ol><li>制备提取介质（每小肠）:30毫升RPMI + 93微升5％（重量/体积）二硫苏糖醇（DTT）+ 60微升的0.5M EDTA +500μl的胎牛血清（FBS）。临用前加入DTT。 </li><li>准备消化介质（每小肠）:25毫升RPMI + 12.5毫克分散酶+ 37.5毫克胶原酶II + 300微升FBS。在使用之前立即加入中性蛋白酶和胶原酶。 </li><li>为在37℃，prewarm溶液至37℃下进行所有温育。 </li></ol></li><li>安乐死用CO 2窒息之后颈脱位鼠标。 </li><li>将鼠标背侧向下和喷雾用70％乙醇的腹部。用剪刀依次切开皮肤，然后腹膜筋膜沿腹中线来回进行剖腹手术m为耻骨联合至剑突过程，从而暴露腹腔。 </li><li>用剪刀由横切幽门括约肌以从胃小肠分开。轻轻地从腹膜取出小肠，挑逗走肠系膜脂肪。 <ol><li>要完全删除小肠，使在回肠盲肠交界处第二晋级。放置分离小肠入冷水（ 即 ，4℃）含有10％FBS的RPMI以最大化细胞生存力。 </li></ol></li><li>轻轻地取出用弯钳大块脂肪。请小心，以避免撕破肠道组织本身，而试图去除脂肪。 </li><li>要除去肠内容物，轻轻冲洗肠，15 - 用固定在一个注射器18G的喂食针20ml冷的PBS。 </li><li>用剪刀切除PPS，并将它们放入含5％FBS冷RPMI。聚苯硫醚位于小肠的系膜侧，并且显示为多叶状白色肿块。根据具体的菌株，鼠标通常具有8 - 12的PP。 </li><li>切小肠进入3 - 4寸段。 </li><li>通过滚动与RPMI浸湿的纸巾各小肠段，用手术刀沉闷挑逗脂肪从组织上卸下剩余的脂肪。 </li><li>通过cannulating与弯钳的肠段与抓取组织的远端转动而外组织。然后用一对直镊子轻轻地去除从弯钳（在近端处开始）的组织段，导致在组织中被反转。 </li><li>将组织细分装有30ml萃取媒介和搅拌棒的杯子。固定在杯子上的盖子，并在37℃下以500rpm搅拌15分钟;搅拌应该是轰轰烈烈，但没有动荡。 </li><li>孵化后，用滤网钢从含有上皮上清液，它应该出现混浊分离组织块。该上清包含了我ntraepithelial淋巴细胞可以进一步分析，如果需要的话，通过进行到过滤在步骤2.18的样品 - 2.23。 </li><li>手动搅动RPMI组织块洗去残留的萃取介质。 </li><li>放在干燥的纸巾组织翻转结束在结束了几次，以便卸下不是由萃取介质解放残留粘液。 </li><li>放置组织片段在1.5ml管600微升的消化培养基。 </li><li>用剪刀将剁碎的组织块，直到不再粘在剪刀和解决方案出现均匀。这个步骤是关键的，以确保组织的完整酶消化。 </li><li>碎小肠添加到含25毫升消化媒体一杯。搅拌在500rpm在37℃下30分钟。半路通过消化（ 即 ，15分钟），吸管上下用血清吸管来帮助打破组织的任何大块。 </li><li>过滤消化的组织（或epith从步骤2.12 elial层如果处理IELs）通过100微米的细胞过滤到50ml管中。冲洗用20ml的RPMI含有10％FBS的过滤器。 </li><li>离心在500 xg离心在4℃下经过滤的溶液10分钟。 </li><li>小心倒出上清，重悬沉淀在1ml的RPMI含有10％FBS。 </li><li>过滤悬浮细胞通过40微米的细胞滤网到50毫升管中。冲洗过滤用20ml的RPMI含有10％FBS。 </li><li>离心机在4℃下过滤溶液，在500×g离心10分钟。 </li><li>小心倒出上清，重新悬浮颗粒在1毫升的RPMI含2％FBS。 </li></ol> 3.单细胞悬液的制备从淋巴集结<ol><li>转移切除百分点至一个杯子用25毫升消化媒体和搅拌棒。安全盖，并在500rpm，10分钟旋在37℃。 </li><li>滤波器消化的PP通过40微米的细胞滤网到50毫升管中。如果有任何团块REMA在使用从1ml注射器柱塞的平端压它们通过过滤器。 </li><li>冲洗过滤器，用10ml的RPMI含有10％FBS。 </li><li>离心机在4℃下过滤溶液，在500×g离心10分钟。 </li><li>小心倒出上清，重新悬浮颗粒在1毫升的RPMI含2％FBS。 </li></ol> 4.表面染色的流式细胞仪<ol><li>细胞的等分试样适当体积（ 例如 ，200微升LPLs的）到96孔圆底板。 </li><li>离心机在500×g离心在4℃下5分钟。通过倒转板倒出上清液。 </li><li>悬浮细胞在90微升含有2％FBS的RPMI中，并以1:100稀释的抗CD16 / 32（Fc的块）。孵育在4℃下10分钟。 </li><li>加10μlPBS。离心机在500×g离心在4℃下5分钟。通过倒转板倒出上清液。 </li><li>悬浮细胞在250微升PBS中。离心机在500×g离心在4℃下5分钟。弃去上清液反相板。 </li><li> （可选的）染色细胞存活率染料，如果需要，通过按照制造商的协议。 </li><li>在100μl1％福尔马林悬浮细胞以固定细胞。孵育在4℃下在黑暗中1小时。 </li><li>加入200μlPBS。离心机在500×g离心在4℃下5分钟。通过倒转板倒出上清液。 </li><li>悬浮细胞在250微升PBS中。离心机在500×g离心在4℃下5分钟。通过倒转板倒出上清液。 </li><li>悬浮细胞在200μlPBS中。分析使用流式细胞仪的细胞。 </li></ol>

<ol>
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No conflicts of interest declared.

我们提出了一个协议，用于分离和流动的小肠淋巴细胞，包括LPLs，IELs，并在PPS淋巴细胞术表征。对于那些有兴趣在评估中微生物的变化如何影响小肠的免疫系统，我们详细介绍参与小鼠窝藏不同microbiotas之间的生物体的水平传播的简单步骤。虽然该协议的重点是小肠，该过程是用于大肠的分析是相同的，唯一的区别是，有没有的PP去除（尽管结肠补丁都存在，这些通常不是严重可见）。 <p class...

小肠的主要任务通常被认为是营养物1的消化和吸收。虽然这种代谢功能显然必不可少的，小肠具有在保护宿主免受内腔2内发现的环境抗原的持续拦河坝同样显著作用。肠道隔开外界（ 例如 ，管腔抗原）从主机的使用本身是只有一个单一的电池层厚的上皮细胞层的内部环境。这样，小肠道免疫系统具有平衡其为反应性的阈值，允许从饮食和共生微生物外来抗原以最小的进入黏膜，如果有的话，免疫反应而安装对抗入侵的病原体和强大的响应的艰巨任务其他"有害的"抗原。到这些抗原过度或不适当的免疫应答可导致病理疾病（ 如 ，inflamma托里肠炎，I型糖尿病，多发性硬化），必须避免3-6。 总体而言，在胃肠道被认为代表在体内最大的免疫器官，含有在所有抗体分泌细胞7的70％。小肠道免疫系统由3个主要的隔层-固有层（LP）时，上皮内层和Peyer氏斑（PPS） -每个包含淋巴细胞2的独特基团。在LP淋巴细胞（LPLs）主要TCRαβ+ T细胞以〜20％的B细胞;上皮内淋巴细胞（IELs）包含极少的B细胞与更多的TCRγδ+ T细胞比TCRαβ+ T细胞;和PPS，它们是嵌入在小肠壁次级淋巴器官，包含〜80％的B细胞。虽然这些解剖区域的稍有不同的功能和本体论基础，他们在发挥作用啊armonized时尚，以防止致病性侮辱的主机。 此外，有越来越多的赞赏菌群对肠道免疫系统的发展的关键因素，随着识别特定的微生物和特定的细胞谱系8,9的个体发育之间的同源关系。此外，鉴于肠道免疫系统的教育影响解剖学远处的免疫应答（ 例如 ，关节炎，多发性硬化症，肺炎），它已经很清楚，在肠道免疫系统的发展是有关多疾病过程比先前识别10 -12。这样，在定量评估肠道免疫系统的兴趣已扩展超出宿主 - 病原体相互作用现在包括主机共生相互作用和许多全身性疾病的发病机制，以及。 定在当前的方法的可变性肠淋巴细胞隔离，即对产量，活力，和一致性优化同时平衡所需要的时间是越来越重要的方法。涉及的Percoll梯度协议是时间和劳动力密集型的，而且可能更容易出现人为错误，导致可变的产量和存活率13。在此，我们提供了淋巴细胞的所有3小肠道免疫车厢分离和鉴定优化的协议。此外，在粘膜免疫系统微生物引起的改变给出越来越大的兴趣，我们包括可用于允许微生物小鼠之间的水平传播，以评估这些变化如何定量影响肠道免疫系统的步骤。

<table>
	<tbody>
		<tr>
			<td>Sterile Gloves</td>
			<td>Kimberly-Clark</td>
			<td>555092</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile mouse cage</td>
			<td>Innovive</td>
			<td>MS2-AD</td>
			<td>contains lid, cage bottom, and alpha-dri bedding</td>
		</tr>
		<tr>
			<td>metal feeder</td>
			<td>Innovive</td>
			<td>M-FEED</td>
			<td></td>
		</tr>
		<tr>
			<td>water bottle</td>
			<td>Innovive</td>
			<td>M-WB-300</td>
			<td></td>
		</tr>
		<tr>
			<td>card holder</td>
			<td>Innovive</td>
			<td>CRD-HLD-H</td>
			<td></td>
		</tr>
		<tr>
			<td>autoclavable rodent chow (NIH-31M)</td>
			<td>Zeigler</td>
			<td>4131207530</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D5545-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>Ambion</td>
			<td>AM9262</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>GemBio</td>
			<td>100-510</td>
			<td></td>
		</tr>
		<tr>
			<td>dispase II</td>
			<td>Invitrogen</td>
			<td>17105-041</td>
			<td>the concentration in the protocol is based on an activity level of 1.878 U/mg</td>
		</tr>
		<tr>
			<td>collagenase, type II&#160;</td>
			<td>Invitrogen</td>
			<td>17101-015</td>
			<td>the concentration in the protocol is based on an activity level of 245 U/mg</td>
		</tr>
		<tr>
			<td>dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>feeding needle (18 G, 2&#34; length)</td>
			<td>Roboz</td>
			<td>FN-7905</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml syringe</td>
			<td>BD</td>
			<td>305482</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel (15 blade)</td>
			<td>Miltex</td>
			<td>4-415</td>
			<td></td>
		</tr>
		<tr>
			<td>curved forceps</td>
			<td>Roboz</td>
			<td>RS-5211</td>
			<td></td>
		</tr>
		<tr>
			<td>straight forceps</td>
			<td>Roboz</td>
			<td>RS-5132</td>
			<td></td>
		</tr>
		<tr>
			<td>multi-purpose cups, 120 ml</td>
			<td>VWR</td>
			<td>89009-662</td>
			<td></td>
		</tr>
		<tr>
			<td>stir bar</td>
			<td>VWR</td>
			<td>58949-062</td>
			<td></td>
		</tr>
		<tr>
			<td>multi-position stir plate, 9-position</td>
			<td>VWR</td>
			<td>12621-048</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel conical strainer, 3 inch&#160;</td>
			<td>RSVP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml tube</td>
			<td>Eppendorf</td>
			<td>0030 125.150</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate, round-bottom</td>
			<td>Corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD16/32 (Fc block)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td></td>
		</tr>
		<tr>
			<td>(optional) fixable viability dye eFluor 780</td>
			<td>eBiosciences</td>
			<td>65-0865-18</td>
			<td></td>
		</tr>
		<tr>
			<td>10% formalin, neutral buffered</td>
			<td>Thermo Scientific</td>
			<td>5725</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and flow cytometric characterization of murine small intestinal lymphocytes

肠子 - 包含在身体任何器官的免疫细胞的数量最多 - 经常暴露于外来抗原，无论是微生物和饮食。鉴于越来越理解是，这些管腔抗原帮助塑造免疫反应和免疫细胞的教育中肠为数字全身性疾病的关键，已经在表征肠道免疫系统的兴趣增加。然而，许多已发表的协议是艰苦和耗时的。我们在这里提出一个简化的协议用于从小肠固有，上皮内层，和淋巴集结是快速的，可重复，并且不需要费力的Percoll梯度淋巴细胞的隔离。虽然该协议着重于小肠，它也适用于结肠的分析。此外，我们突出一些方面中，可能需要额外的优化取决于特定科学疑问句化。这种方法导致了大量可行的淋巴细胞，可以随后用于流式细胞术分析或表征备用装置的隔离。

流的小肠淋巴细胞的单细胞悬浮液的流式细胞仪分析应该产生具有向前相似和侧向散射特征的脾细胞（ 图1A和1B）细胞的离散人口。淋巴细胞可开始死亡如果在隔离的初始阶段的组织不保持在4℃，导致淋巴细胞群具有较低的前向散射和是更难以从其他上皮细胞和死细胞分离（ 图1C） 。此外，如果小肠组织没有被完全清洁的其肠系膜脂肪，有实质?...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes at JoVE.com

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

isolation-flow-cytometric-characterization-murine-small-intestinal

Research

JoVE Journal

Immunology and Infection

27.3K 次观看.  Boston Children’s Hospital. There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization. 

视频: Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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 Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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

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

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

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

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

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

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 and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.