Name: isolation and flow cytometric characterization of murine small intestinal lymphocytes
Uploaded: 2016-05-08T15:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes at JoVE.com

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>

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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&amp;nbsp;</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&quot; 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&amp;nbsp;</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 &amp;mu;m Cell strainer</td><td>Falcon</td><td>08-771-19</td><td></td></tr><tr><td>40 &amp;mu;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

27.4K 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

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 ...

科研

免疫与感染

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 ...

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 ...

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 ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

流式细胞术测定小鼠胸腺、胰腺干淋巴结和脾脏的调节 t 细胞亚群

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

小鼠B细胞发育的流式细胞学表征

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

分离子宫先天淋巴细胞，用于流式细胞术分析

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

小鼠小肠上皮类器官与先天性淋巴细胞的共培养

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

生物学

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的 体内 动力学

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...

小鼠小肠淋巴细胞的分离和流式细胞表征

摘要

探索更多视频

此视频中的章节

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

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

分离及小鼠淋巴细胞的激活

小鼠小肠免疫系统淋巴细胞的分离

流式细胞术测定小鼠胸腺、胰腺干淋巴结和脾脏的调节 t 细胞亚群

小鼠B细胞发育的流式细胞学表征

分离子宫先天淋巴细胞，用于流式细胞术分析

小鼠小肠上皮类器官与先天性淋巴细胞的共培养

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的体内动力学

小鼠小肠淋巴细胞的分离和流式细胞表征

摘要

探索更多视频

此视频中的章节

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

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

分离及小鼠淋巴细胞的激活

小鼠小肠免疫系统淋巴细胞的分离

流式细胞术测定小鼠胸腺、胰腺干淋巴结和脾脏的调节 t 细胞亚群

小鼠B细胞发育的流式细胞学表征

分离子宫先天淋巴细胞，用于流式细胞术分析

小鼠小肠上皮类器官与先天性淋巴细胞的共培养

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的 体内 动力学

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的体内动力学