我们感谢亚伦RAE（埃默里大学儿科和儿童医疗亚特兰大流核心），细胞分选。这项工作是由NIH资助AA01787001，从克隆氏病及溃疡性结肠炎美国基金会事业发展奖，和埃默里Egleston儿童研究中心种子批支持到顶级域名（TLD）

1。肠上皮细胞的解剖和离解制备的试剂和设备: <ul><li>温暖的Ca 2 + /镁2 +无PBS（CMF的PBS）的室温。 </li><li>温暖的Ca 2 + / Mg 2 +的免费的HBSS 5％胎牛血清（CMF方案的HBSS / FBS），和2mM EDTA室温。 </li><li>温暖的轨道摇床至37°C。 </li></ul> 注:步骤1.1至1.7，必须尽快进行，以尽量减少细胞死亡的程度，并实现最大的细胞产量。 <ol><li>安乐死小鼠中的CO 2室及喷到腹部和胸部70％的乙醇。 </li><li>让小剪刀和剥离皮肤暴露腹膜横切口在腹部中间。 </li><li>从小肠上部进行分离胃幽门括约肌切割。戏弄了用钳子肠系膜和再次削减回肠盲肠的阀门释放整个小肠，大肠。使肛门边缘的切割和再次捉弄除了肠系膜，直到大肠是免费的。 </li><li>剖开结肠，纵向用剪刀在CMF的PBS在室温下肠腔内粪便的内容和粘液洗掉。 </li><li>用剪刀和镊子宏观剖析反肠系膜表面沿小肠Peyer氏斑和纵向剖开小肠。 </li><li>在室温下洗小肠管腔粪便的内容和粘液在CMF的PBS。 </li><li>分别含30毫升预热的CMF方案的HBSS /胎牛血清和2mM EDTA单独的50毫升锥形管小/大肠切成约1.5厘米的块和地方。不要添加超过50毫升锥形管的1％的小肠。 </li><li>每50毫升锥形管水平放入轨道摇床和在250转摇在37°C &lt;20分钟/ LI&gt; <li>废物桶置于一个单一的铁丝网过滤，倒入每次50毫升锥形管的内容，通过收回1.5厘米的小肠件，并将其放置在独立50预的温暖CHBSS /胎牛血清30毫升含2毫升锥形管mM的EDTA。 </li><li>重复1.8。 </li></ol> 2。组织消化和肠细胞的分离试剂和设备的准备: <ul><li>胶原酶溶液: 1.5毫克/毫升的类型第八胶原酶溶解在CMF的HBSS中预温/胎牛血清与40微克/毫升的DNase I。 </li><li> CMF的HBSS中预温/胎牛血清和2mM EDTA。 </li><li>预热的轨道摇床在37°C </li><li>冰冷的CMF方案的HBSS / FB的。 </li></ul><ol><li>晃动的第二轮结束后，通过过滤网倒入每次50毫升锥形管的内容和转移1.5厘米的小肠一个小塑料件重量船后，涂抹了多余的介质，使用纸巾。 </li><li>迅速切末1.5厘米小肠件重量船直接用剪刀和胶原酶溶液20毫升加入剁碎的小肠。每50毫升锥形管放入轨道摇床和消化水平在10-20分钟200转37°C. 请参见下面有关优化的讨论。 </li><li>简要涡通过100μm的细胞过滤器，直接到50毫升锥形管，以确保任何剩余的肠道组织和过滤器的彻底分离。 </li><li>关闭每50毫升锥形管的顶端与CMF方案的HBSS / FB和1500转离心5分钟，4°C。如果固体颗粒不离心后观察结肠样品，样品应为3.5分钟再次离心。再次重复此清洗步骤。 </li><li>倒掉上清，重悬细胞沉淀在冰冷的CMF方案的HBSS / FBS和地方样本上冰。 </li><li>进行流式细胞仪采集/分析或磁珠富集高速细胞sorti的第4条第3节纳克。 </li></ol> 3。多色流区议会和巨噬细胞的流式细胞仪分析抗体染色试剂和设备的准备: <ul><li>冰冷CMF的PBS。 </li><li>冰冷的染色缓冲（CMF的PBS + 5％FBS）。 </li><li>准备在1:1000稀释死细胞染色，用活/死可修复水族死细胞染色试剂盒，在冰冷的CMF的PBS。 </li><li>准备加入荧光标记单克隆抗体（mAb）冰冷的染色缓冲抗体染色鸡尾酒:CD45-PerCP，CD103-PE，CD11c的，APC，MHC-Ⅱ（执行机构）的Alexa Fluor 700，CD11b的eFluor 450，F4/80-PE-Cy7。 </li></ul><ol><li>转移到5毫升的聚苯乙烯圆底（流式细胞仪）管细胞。 </li><li>在冰冷的CMF的PBS洗涤细胞两次。 </li><li>染色死细胞孵育15分钟就在黑暗中的冰。 </li><li>在冰冷的CMF的PBS洗涤细胞两次。 </li><li> 2.4G2anti-FcγRIII/II座在冰冷的染色细胞在冰上10分钟的缓冲。 </li><li>在冰冷的染色缓冲洗细胞。 </li><li>孵育抗体染色鸡尾酒在黑暗中冰20分钟的样品。 </li><li>与冰冷的染色缓冲洗细胞两次，并悬浮在400μl冰冷的染色缓冲区的样品，并通过流式细胞仪管40微米的过滤器盖。 </li><li>收购上的LSR II流式细胞仪（BD）作为由浇注战略定义，在第5和图1的样品。 </li></ol> 4。富集区议会和巨噬细胞从小肠试剂和设备的准备: <ul><li>冰冷的染色缓冲（CMF的PBS + 5％FBS）。 </li></ul><ol><li>孵育根据制造商的指示，从步骤2.5以CD11b和CD11c磁珠珠获得单细胞悬液。 </li><li>用离心缓冲区冰冷的染色细胞。 </li><li>弃上清，悬浮细胞沉淀用1 mL冰冷的染色缓冲并通过细胞由40微米的细胞过滤过滤100微米。 </li><li>磁珠连接细胞丰富，通过积极选择使用磁珠的LS磁列。 </li><li>重复步骤4.2和弃上清。 </li><li>孵育步骤3.7中所述的表面标记单克隆抗体的细胞。 </li><li>洗两次磁珠富集细胞与冰冷的染色缓冲。重悬细胞沉淀，在500μL冰冷无叠氮化钠染色缓冲，并通过40微米的细胞过滤器传递到流式细胞仪管。 </li><li>进行屋宇署唱腔二，细胞分选，排序肠道DC和/或利益的巨噬细胞亚群流式细胞仪分选。 </li></ol> 5。浇注战略为LP装甲运兵车 注 :请注意，未染色的肠细胞可能被利用作为阴性对照，以协助妥善安置门正面和负面的人群分开。 <ol><li> 如图1，创建一个点图和排除细胞是死细胞染色（ 图1A），其次是排斥的双重事件（ 图1B和C）呈阳性反应。然后，根据转发侧分散决策一定要排除碎片（ 图1D）的利息细胞的大门。 </li><li>创建另一个点的情节和CD45的进一步门+和IA B +细胞，表型特征装甲运兵车（ 图1E）。 </li><li>在一个单独的点图，分析CD11b和CD11c的表达来区分特定的DC和巨噬细胞亚群（R1，R2和R3。图1F）。 R1的细胞素CD11c + CD11b的沉闷/ -细胞。地区，R2被划定为CD11c的，这些细胞有R1细胞表面表达水平相似，但R2细胞也表达CD11b的。此后，R3的地区被指定的细胞，细胞CD11b +和CD11c 沉闷/ - 。 </li><li>一个alyze地区R1，R2和R3的进一步F4/80的和CD103表达分化巨噬细胞和DC人群，分别。在素CD11c + CD11b的沉闷/ - αE整合，CD103，F4/80的较低水平（ 图1G）R1的快递高水平的细胞。细胞CD11b + CD11c的+ R2的基于二元表达CD103和F4/80的（ 图1H）两个区议会和巨噬细胞组成的细胞。最后，细胞CD11b + CD11c的沉闷/ - R3的细胞构成基于表型剖面的F4/80的+和CD103的巨噬细胞- （ 图1I）16。 </li></ol> 6。代表结果<img alt="图1" src="/files/ftp_upload/4040/4040fig1.jpg" /> 图1。首先从分析中排除浇注战略肠道DC和巨噬细胞，死细胞（A）和双峰（B和C），然后小intestiNAL细胞门相应的转发和侧散射（四），装甲运兵车被定义为CD45的保险业监督B +（E）巨噬细胞和DC的CD11b和CD11c（F）的表达。 CD103细胞预门R1（七），R2（H）和R3（一）人口和F4/80的表达进行了分析。 <img alt="图2" src="/files/ftp_upload/4040/4040fig2.jpg" /> 图2。细胞产量和抗体染色的质量取决于消化时间。CD11b和CD11c的染色模式下（D）总细胞产量，最佳（乙，戊类）或过度消化（，F）的肠道组织。 从C57BL / 6小鼠小肠肠细胞的分离和区议会和巨噬细胞上的BD LSR II流式细胞仪分析。使用未染色和荧光染色脾设置电压和补偿。首先排除死细胞（ 图1A）和双峰（ 图1B和C）分析。感兴趣的细胞，然后根据转发和分析，其次对CD45的门侧散射（ 图1D）+和IA B +细胞（ 图1E）。此后，CD11b和CD11c的表达评估之间的CD45 +保险业监督B +细胞划定三个区域（R1，R2和R3。图1F）。评估，区议会和巨噬细胞之间的区分，分别在三个区域中CD103和F4/80的表达。在素CD11c + CD11b的沉闷/ - R1细胞表达高水平αE整合，CD103，F4/80的较低水平（ 图1G）。细胞CD11b + CD11c的+ R2的细胞组成的基于二元表达CD103和F4/80的（ 图1H）两个区议会和巨噬细胞，而细胞CD11b + CD11c的沉闷/ - R3的细胞构成/ F4的表型剖面的基础上的巨噬细胞80 +和CD103 - （ <strONG&gt;图。 1I）16。在R2的门的巨噬细胞和巨噬细胞中的R3门也有类似的正向和侧面散射特性和CD11c的表达区分。这些亚群功能的二分法仍然不完全理解。 该组织总细胞产量和细胞CD11b和CD11c表达的消化时间之间的关系如图2所示。 3分钟消化（消化下）肠组织产生了较低的总细胞数（ 图2D），因此，很少的区议会和巨噬细胞的特性（ 图2A）。组织11分钟的消化产生了强劲的活细胞（ 图2E）产量与人口的区议会和巨噬细胞表达高水平的CD11b和CD11c和表型不同（ 图2C）。相比之下，在一个类似的细胞产量为50分钟（超过消化）消化导致当合作mpared优化消化（ 图2E和F），然而，划定不同的细胞，利用细胞CD11b和CD11c人口成为表达CD11c的减少更晦涩难懂（ 图2C）和死细胞数量增加（数据未显示）。

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++Th+cells+resembling+regulatory+T+cells+that+inhibit+chronic+colitis+differentiate+in+the+absence+of+interactions+between+CD4+and+class+II+MHC.">CD4+ Th cells resembling regulatory T cells that inhibit chronic colitis differentiate in the absence of interactions between CD4 and class II MHC.</a> J. Immunol. 171, 2279-2286 (2003).</li></ol>

没有利益冲突的声明。

<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="图3"/> 图3。直接组织消化时间的影响因素，重要的细胞产量和表面抗原表达的优化。细胞产量和表面抗原表达，胶原酶的具体特点，组织切碎程度，以及存在或炎症的情况下，这可能会影响组织的完整性和细胞结构。长期组织消化，可能会导致细胞活力下降和表面抗原的表达，同时组织消化不足可能会导致在分析细胞缺乏。 ...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 1×PBS的Ca 2 + -和Mg 2 +无</td><td></td><td></td><td></td></tr><tr><td>汉克的平衡盐溶液（HBSS中）与酚红</td><td> Fisher Scientific则</td><td> SH3001603 </td><td></td></tr><tr><td>碳酸氢钠</td><td>西格玛</td><td> S6014 </td><td></td></tr><tr><td>在0.85％的NaCl 1M肝素钠</td><td>龙沙</td><td> 17-737E </td><td></td></tr><tr><td>胎牛血清（FBS）， </td><td>亚特兰大生物制品</td><td> S11150H </td><td>热灭活</td></tr><tr><td> 0.5M EDTA的（pH值8.0） </td><td> cellgro </td><td> 46-034-CI </td><td></td></tr><tr><td> collagenasE型八</td><td>西格玛</td><td> C2139 </td><td></td></tr><tr><td> DNA酶I </td><td>罗氏公司</td><td> 14785000 </td><td>联合解决方案:100mg/ml </td></tr><tr><td> LIVE / DEAD可修复水族死细胞染色试剂盒，为405 nm激发</td><td> Invitrogen公司</td><td> L34957 </td><td>使用1:1000 </td></tr><tr><td> CD45 - ，PerCP单克隆抗体（30F11） </td><td>屋宇署</td><td> 557235 </td><td>使用1:100 </td></tr><tr><td> CD103-PE单克隆抗体（M290） </td><td>屋宇署</td><td> 557495 </td><td>使用1:100 </td></tr><tr><td> FcγRIII/ II抗体（2.4G2） </td><td>屋宇署</td><td> 553141 </td><td>在1:200的使用</td></tr><tr><td> CD11c的APC的单克隆抗体（N418） </td><td> eBioscience </td><td> 17-0114-82 </td><td>使用1:100 </td></tr><tr><td> MHC-Ⅱ（我-AB）的Alexa Fluor 700单克隆抗体</td><td> eBioscience </td><td> 56-5321-82 </td><td>使用1:100 </td></tr><tr><td>细胞CD11b-eFluor 450单克隆抗体（M1/70） </td><td> eBioscience </td><td> 48-0112-82 </td><td>在1:200的使用</td></tr><tr><td> F4/80-PE-Cy7单克隆抗体（BM8） </td><td> eBioscience </td><td> 25-4801-82 </td><td></td></tr><tr><td> CD11b的微球</td><td>美天旎生物技术</td><td> 130-049-601 </td><td></td></tr><tr><td> CD11c的微球</td><td>美天旎生物技术</td><td> 130-052-001 </td><td></td></tr><tr><td> 50 ml锥形管</td><td>屋宇署猎鹰</td><td> 352098 </td><td></td></tr><tr><td>单铁丝网过滤</td><td> Chefmate </td><td></td><td></td></tr><tr><td>小重量船</td><td> Fisher Scientific则</td><td> 08-732-116 </td><td></td></tr><tr><td> 100微米的细胞过滤器</td><td>屋宇署猎鹰</td><td> 352360 </td><td></td></tr><tr><td> 40微米的细胞过滤器</td><td>屋宇署猎鹰</td><td> 352340 </td><td></td></tr><tr><td> 5毫升聚苯乙烯圆底管</td><td>屋宇署猎鹰</td><td> 352235 </td><td>使用1:100 </td></tr><tr><td> MAXQ 4450台式轨道摇床</td><td> Thermo Scientific的</td><td></td><td></td></tr><tr><td>的LS磁珠列</td><td>美天旎生物技术</td><td> 130-042-401 </td><td></td></tr><tr><td> LSR的二</td><td>屋宇署</td><td></td><td></td></tr><tr><td> FACSAria二</td><td>屋宇署</td><td></td><td></td></tr></tbody></table>

isolation and characterization of dendritic cells and macrophages from the mouse intestine

肠道内居住的是参与对共生的植物和食物抗原在促进容忍，而随之而来的剩余准备安装的炎症反应，病原体侵入朝1,2先天免疫和适应性免疫细胞的独特群体。抗原提呈细胞，特别是区议会和巨噬细胞，发挥关键作用，在肠道免疫稳态维持通过自己的能力感和妥善应对微生物3-14。肠道DC和巨噬细胞的高效分离是在描述这些细胞的表型和功能的关键一步。虽然隔离肠道免疫细胞，包括区议会和巨噬细胞，许多有效的方法已6,10,15-24，许多依靠长期的消化时间，可能会产生负面影响细胞表面抗原的表达，细胞活力，和/或细胞产量。在这里，我们详细介绍快速分离的大量viabl的一种方法E，肠区议会和巨噬细胞。肠道DC和巨噬细胞的表型特征进行了直接染色孤立的肠细胞与特定的荧光标记单克隆抗体多色流式细胞仪分析。此外，高纯度的DC和巨噬细胞群体隔离功能的研究，利用CD11c和CD11b的磁激活细胞分选，细胞分选微珠。

Watch this Scientific Journal Video about Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine at JoVE.com

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

在这里，我们详细介绍了小鼠肠道树突状细胞（DCs）和巨噬细胞的快速分离方法。使用多色流式细胞仪分析细胞分选磁珠富集而被用来产生高纯度的功能研究人口肠道DC和巨噬细胞的表型特征。

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-characterization-dendritic-cells-macrophages-from-mouse

Research

JoVE Journal

Immunology and Infection

39.0K 次观看.  Emory University. 在这里，我们详细介绍了小鼠肠道树突状细胞（DCs）和巨噬细胞的快速分离方法。使用多色流式细胞仪分析细胞分选磁珠富集而被用来产生高纯度的功能研究人口肠道DC和巨噬细胞的表型特征。

视频: Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

科研

免疫与感染

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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 ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.