的作者想感谢张冰，他的技术援助。这项工作是由加拿大卫生研究院的研究（MOP-86595）。 TAB是一个CIHR的新研究者和AHFMR学术。 QH的支持，一个CIHR加拿大研究生奖学金 - 博士和一个AIHS的全日制学籍。 SAN是由英国女王伊丽莎白二世研究生奖学金的支持。 AYWS支持由NSERC - 博士研究生奖学金。

请参阅图1为实验方案的总体方案。 1。解剖<ol><li>地方无菌钢丝网屏幕为60 x 15 mm的培养皿中。需要一个单元的每个组织样本。 </li><li>每个培养皿中加入5ml的Hank氏平衡盐溶液（HBSS）。保持在冰上菜。 </li><li>安乐死小鼠与CO 2。 </li><li>安全小鼠剥离面，腹一面朝上。用70％乙醇进行杀菌，并确保皮草纠结向下喷雾鼠标。 </li><li>用手术剪，开始清扫腹切口在腹部以上的生殖器皮肤。向上延长切口到下巴。 </li><li>从中线，延长切口，沿四肢。将松弛的皮肤和针剥离面。 </li><li>收获的胸腺： <ol><li>做一个切口胸骨抬起底尖。</li><li>避免肝脏，切取下的肋骨，然后切肋骨两侧各隔膜。在每边，切肋骨向上照顾，以避免肺和心脏。 </li><li>轻轻地拉钳胸腔回来。胸腺是白色的，双叶器官的心脏上方。使用的钳子抓住底部的叶平边，轻轻一拉，胸腺，并把它放在丝网的目数。 </li></ol></li><li>收获脾： <ol><li>脾是一个红色，冲浪板形器官位于左侧的老鼠的腹腔内，下面的肝脏上。 </li><li>做一个切口进入腹腔。一个钳子轻轻拉出脾，使用第二对逗结缔组织的。放置在一个单独的目筛脾。 </li></ol></li></ol> 2。细胞制备<ol><li>使用从3毫升注射器的柱塞，研磨成网状屏幕上，直到只有结缔组织和脂肪的遗体器官。冲洗啮合HBSS中从盘三次。每个组织样本，使用一个新的柱塞。 <ol><li>代替这种方法可以用在其他可供选择的均质组织。 </li></ol></li><li>使用血球计数仪计数细胞。 </li><li>通过离心沉淀细胞在335×g离心5分钟，在4℃下</li><li>重悬浮的脾细胞在500μl的ACK裂解缓冲液（0.15M NH 4 Cl的 ，10 mM的KHCO 3，0.1mM的的Na 2 EDTA，pH7.2）中，在室温下的10分钟。重悬的胸腺细胞，在20×10 6细胞/ ml在FACS缓冲液（PBS，1％FCS，0.02％叠氮化钠），并设置预留在冰上。 </li><li>返回通过加入5毫升的HBSS中的脾细胞对等渗。佩莱脾细胞通过离心分离和重新悬浮在20×10 6细胞/ ml在FACS缓冲液中。如果细胞需要保持无菌状态，您可以使用无菌RPMI + 10％FCS。 </li></ol> 3。染色细胞流式细胞仪此协议的目的是，勾勒战略的分析选择使用野生型（WT）和HY CD4小鼠的正面和负面的。流式细胞仪的实验设计，采集，和分析的一般注意事项，请读者一个很好的审查由东等人。 <ol><li>等分试样4×10 6个每流式细胞仪样品的胸腺细胞的96孔板的各孔，以及1×10 6个 WT脾细胞每补偿控制。如果使用转基因小鼠中，应该包括一个野生型小鼠作为对照在你的实验。 </li><li>座和10分钟，在冰上孵育细胞与anti-CD16/32（克隆2.4G2）的Fc受体。 </li><li>附带板上，在335×g离心5分钟，在4℃下。取下盖子，消除井液体从弹板后，面朝下放入水槽。重悬于200μl的FACS缓冲液中的各孔中。重复洗涤。 </li><li>制备抗体鸡尾酒，列出如下，使用基于稀释于200μlFACS缓冲液，每孔各抗体的最佳浓度。每种抗体的最佳浓度是指，得到最大的分离阳性和阴性的人群所需的最低浓度。如果没有对于一个给定抗体的人口负，应包括合同种型抗体。每孔的总体积可以缩小（ 例如 ，以每孔100μl），如果需要的话。 <ol><li> WT胸腺：抗-TCRβ，抗-CD4，抗-CD8，抗CD69或抗CD5，抗-CD24 </li><li> HY CD4胸腺：抗-HY TCR（T3.70），抗-CD4，抗-CD8，抗CD69或抗CD5，抗-CD24 为了获得更好的分离的CD69 lo和CD69 您好种群在胸腺细胞，它是推荐的，可以使用生物素化的抗CD69初级抗体，随后的二次染色含有荧光标记的链霉抗生物素蛋白。我们üCD5染色本质上相同的策略。 </li></ol></li><li>培养细胞的抗体鸡尾酒用200μlFACS缓冲液在冰上30分钟，在黑暗中。 </li><li>同时，抗体鸡尾酒染色，染色每个补偿控制一个单一的荧光标记的抗体。在理想的情况下，补偿染色涉及抗体缓冲液中所用的相同的抗体。然而，为每个单独的抗原染色可能不放大的实验中是可行的许多抗原时正在测定。因此，任一抗-CD4或抗-CD8染色建议由于这些抗原高表达，或使用的补偿珠。染色FACS缓冲30分钟冰在黑暗中。保持一个补偿控制不染。 </li><li>用FACS缓冲液洗涤细胞两次。 </li><li> FACS缓冲液悬浮细胞在转移到FACS管。 </li><li>流式细胞仪采集样品。包括收购的FSC-A和FSC-W到llow双重歧视。 </li></ol> 4。流式细胞仪数据分析 - 非TCR转基因小鼠我们使用FlowJo流式细胞仪数据分析。请参阅图2A为选通非TCR转基因小鼠的策略。 <ol><li>使用FSC-A，SSC，电子门上的“细胞”人口。 </li><li>在“细胞”的人口，使用FSC-A由FSC-W到电子门的FSC-W LO人口，排除细胞的双峰和aggregrates的。这是“单”门。 </li><li>使用“单”门的CD4 CD8，情节的事件。绘制门为DN DP，DP 沉闷 ， 明亮 ，CD4 + CD8 不料 ，CD4SP和的CD8SP人口为描绘在图2B。 </li><li>根据CD4SP和CD8SP门，创造的TCRβCD24地块。使用的象限门控工具绘制图2C所示的门。 </li><li>创建一个NE在瓦特情节从“单门：TCRβCD69。绘制门A，B，C，D，如在图2D中所示。 </li><li>从“单门：TCRβCD5创建另一个情节。绘制门I，II，III，IV所示， 图2E，这是另一种战略研究正选择。 </li><li>从“单”应用DN，DP 平淡 ，DP 明亮 ，CD4 诠释 ，CD4SP和CD8SP门门I，II，III，IV，A，B，C，D，E（图2D）。 </li><li>计算细胞的数量乘以器官的细胞结构，后续的每一个门的频率，直到您达到您的目标人群中的特定子集。例如，TCRβ 喜 CD69-CD8SP胸腺细胞的数量将取决于胸腺细胞结构的x％TCR BHI CD69（部分D）x％的CD8SP。 <ol><li>计算已考虑到活细胞和死细胞，所以不包括“淋巴细胞&gt;YTE“门在你的计算。 </li></ol></li></ol> 5。流式细胞仪数据分析 - TCR转基因小鼠请参阅图3A为TCR转基因小鼠的选通战略。 <ol><li>在上一节，按照步骤4.1和4.2。 </li><li>在“单”门“中，创建一个T3.70 SSC的情节和门上的T3.70 +细胞群体分析抗原特异性T细胞（ 图3B）。在某些情况下，它可能会感兴趣的比较人口非抗原特异性（T3.70-）T细胞群体。 </li><li>在T3.70 +人口，抽奖DN，DP，CD4SP和CD8SP门（ 图3C）。 <ol><li> WT对照样品，这些门画下的“单”门创建一个T3.70门，会有极少数T3.70 +细胞。 </li></ol></li><li>在的CD8SP门，创建一个T3.70的CD24情节。使用象限门控工具来将细胞分为四个群体（ 图3F）。 </li><li>创建一个叠加的直方图描述CD69的表达野生型小鼠的DP盒和T3.70 + DP室HY CD4小鼠（ 图4A）。重复检查CD5的表达（ 图4B）。 </li><li>计算在每个子集的兴趣细胞的绝对数量，如4.8。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+analysis+of+clonal+deletion+in+vivo.">Transcriptional analysis of clonal deletion in vivo.</a> J. Immunol. 179, 837-844 (2007).</li></ol>

没有利益冲突的声明。

这里提出的协议可以被用来检查非TCR转基因和TCR转基因小鼠中的阳性和阴性选择。此协议描述了表面抗原染色。有关的分子机制的进一步的分析，这是经常需要进行细胞内染色。我们使用了BD Biosciences公司的Cytofix / Cytoperm工具包的大多数细胞内的蛋白质和BD Biosciences公司的Foxp3的染色试剂盒的转录因子。通常，我们获得了我们的样品后，立即染色。然而，样品可以被存储在FACS缓冲液中过夜用1％多?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> HyClone公司的Hank平衡盐溶液</td><td> Thermo Scientific的</td><td> SH30030.02 </td><td></td></tr><tr><td>金属网帘</td><td> CEDARLANE </td><td> CX-0080-E-01 </td><td></td></tr><tr><td>皮氏培养皿（60×15毫米） </td><td> Fisher Scientific则</td><td> 877221 </td><td></td></tr><tr><td>注射器（3毫升） </td><td> BD Biosciences公司</td><td> 309657 </td><td></td></tr><tr><td>锥形管（15毫升） </td><td>萨尔斯塔特</td><td> 62.554.205 </td><td></td></tr><tr><td>显微镜</td><td>蔡司 - 普里莫之星</td><td> 415500-00XX-000 </td><td></td></tr><tr><td>血球计数仪</td><td>豪塞尔科学的</td><td> 3110 </td><td></td></tr><tr><td> 96 - 孔板</td><td>萨尔斯塔特</td><td> 82.1582.001 </td><td></td></tr><tr><td>多道移液器</td><td> Fisherbrand </td><td> 21-377-829 </td><td></td></tr><tr><td>胎牛血清的</td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td>磷酸盐缓冲盐水</td><td> Fisher Scientific则</td><td> SH3025802 </td><td></td></tr><tr><td>叠氮化钠</td><td> IT贝克化工有限公司</td><td> V015-05 </td><td></td></tr><tr><td> Fc受体阻断剂</td><td></td><td></td><td>克隆2.4G2 </td></tr><tr><td> ANTI-MOUSE HY TCR </td><td> eBioscience公司</td><td> XX-9930-YY * </td><td>克隆T3.70 </td></tr><tr><td>抗小鼠CD4 </td><td> eBioscience公司</td><td> XX-0042-YY * </td><td>克隆RM4-5 </td></tr><tr><td>一NTI鼠标CD8α </td><td> eBioscience公司</td><td> XX-0081-YY * </td><td>克隆53-6.7 </td></tr><tr><td> ANTI-MOUSE CD24 </td><td> eBioscience公司</td><td> XX-0242-YY * </td><td>克隆M1/69 </td></tr><tr><td>抗鼠TCRβ </td><td> eBioscience公司</td><td> XX-5961-YY * </td><td>克隆H57-597 </td></tr><tr><td> ANTI-MOUSE CD69生物素</td><td> eBioscience公司</td><td> 13-0691-YY * </td><td>克隆H1.2F3 </td></tr><tr><td>抗鼠CD5生物素</td><td> eBioscience公司</td><td> 13-0051-YY * </td><td>克隆53-7.3 </td></tr><tr><td>链霉亲和素</td><td> eBioscience公司</td><td> XX-4217-YY * </td><td></td></tr><tr><td>流式细胞仪</td><td> BD Biosciences公司 - FACS坎托</td><td> 338962 </td><td></td></tr><tr><td> FACS管</td><td> BD Biosciences公司</td><td> 352052 </td><td></td></tr><tr><td>流式细胞仪分析软件</td><td> TreeStar - Flowjo </td><td> FlowJo V7 / 9 </td><td></td></tr><tr><td>胎牛血清RPMI - 1640 </td><td> Thermo Scientific的</td><td> SH30027.01 </td><td></td></tr><tr><td></td><td></td><td></td><td> * XX不同的荧光染料和YY的瓶大小的变化而变化。 </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

健康的免疫系统需要对外来抗原的T细胞反应，而其余的宽容，以自身抗原。随机重排的T细胞受体（TCR）的α和β位点产生一个具有丰富的多样性，在抗原特异性T细胞库，无论是对自己和国外。安全和有用的T细胞在胸腺发育过程中的曲目的选择是至关重要的。胸腺选择的缺陷的发展，自身免疫性疾病和免疫缺陷病的1-4。 T细胞祖细胞进入胸腺双阴性（DN）胸腺细胞不表达CD4或CD8共受体。 αβTCR和两个共受体的表达发生在双阳性（DP）阶段。互动的自我肽-MHC（住房抵押贷款公司）提出的胸腺细胞的αβTCR确定DP胸腺细胞的命运。高亲和力相互作用导致负选择和消元重刑自反应的胸腺细胞。在积极的选择和发展的CD4或CD8单阳性（SP）T细胞能够识别外来抗原的自我MHC 5低亲和力相互作用的结果。 的阳性筛选可以研究在小鼠的多克隆（野生型）的TCR剧目通过观察成熟T细胞的产生。然而，他们研究的阴性选择，这涉及到删除的小抗原特定人群的理想选择。许多模型系统已被用于研究的阴性选择，但不同的能力复述的生理活动6。例如， 在体外刺激的胸腺细胞缺乏选择是密切参与胸腺环境，而施用外源性抗原可导致非特异性缺失胸腺细胞7-9。目前，最好的工具，研究在体内的负选择，表达一个transg的有老鼠ENIC TCR特异的内源性自身抗原。然而，许多经典的TCR转基因模型的特点是在DN阶段的过早表达的转基因TCRα链，导致过早的负选择。我们的实验室已经开发出了HY的CD4模型，其中转基因HY，TCRα是有条件表示在自行分配学位阶段，让阴性选择过程中发生的DP，SP转型发生在野生型小鼠10。 在这里，我们描述了一个基于流式细胞仪的协议在HY CD4小鼠模型，研究胸腺的阳性和阴性选择。虽然负选择HY CD4小鼠中是高度生理，这些方法还可以被应用到其他的TCR转基因模型。我们也将目前的一般策略分析正选择一个适用于任何转基因操作的小鼠多克隆剧目。

在生理TCR转基因模型和野生型小鼠，正开始选择在DP 明亮的舞台前，后移动到DP 沉闷的阶段抗原相遇。 DP 沉闷的胸腺细胞，然后进入一个过渡CD4 + CD8 罗的舞台前，成为CD4SP或CD8SP胸腺细胞（ 图2B）。成熟的SP胸腺细胞的特点是高的TCR表达和损失的CD24（ 图2C）。虽然CD8 CD4配置文件可以发现缺陷，积极的选择，检查TCRβCD69或CD5可以提?...

Watch this Scientific Journal Video about Examination of Thymic Positive and Negative Selection by Flow Cytometry at JoVE.com

Examination of Thymic Positive and Negative Selection by Flow Cytometry

我们提出了一种基于流式细胞仪的方法来研究T细胞发育<em在体内</em使用基因操纵的野生型小鼠的T细胞受体的转基因背景。

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

examination-thymic-positive-negative-selection-flow

Research

JoVE Journal

Immunology and Infection

21.6K 次观看.  University of Alberta. 我们提出了一种基于流式细胞仪的方法来研究T细胞发育

视频: Examination of Thymic Positive and Negative Selection by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

在啮齿动物疟原虫原虫的体内评估和裂殖子入侵流式细胞仪

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

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 ...

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.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

双氟铬流式细胞仪对七种免疫细胞子集的判别

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...