我们要感谢他在使用流式细胞仪的核心支持条例草案Cashdollar博士。

<ol><li>开始之前的过程中，下列项目需要收集和准备： <ol style="list-style-type:lower-roman;"><li>冰</li><li> 15毫升锥形管染色细胞和染色单一的颜色控制的12 × 75毫米流量管。 </li><li>染色缓冲液：磷酸盐缓冲液（PBS）+ 3％小牛血清（FCS） </li><li>移液器</li><li> FC块（如果需要）和染色抗体。最佳染色抗体需要滴定。 </li><li>补偿珠</li><li>暂停缓冲：汉克的平衡盐溶液（HBSS）+ 25毫米的HEPES + 3％FCS </li><li>台盼蓝和血球</li><li>细胞过滤器（40微米尼龙） </li><li>收集管：两种类型的收集管，可用于A）12 × 75毫米的聚苯乙烯管（含300μLFCS和25毫米的HEPES）或b）15毫升（含1毫升的FCS + 25毫米的HEPES），锥形管。排序细胞的集合，可用于任何具有丰富的血药浓度高媒介。 </li></ol></li><li>生成一个起始细胞群的单细胞悬液。 </li><li>可选：充实所需的细胞群，批量纯化方法作为补充枯竭或磁性排序等。浓缩步骤的主要优点是，它减少了排序时间。 </li><li>与染色缓冲液洗涤细胞一次。 </li><li>弃上清，重悬细胞染色浓度的缓冲区，高效染色50 × 10 6。 </li><li>可选：细胞表达高水平的FCR，使用适当的阻塞方法阻止受体。一个选择的方法是使用单克隆抗体结合FcγR冰10-15分钟。这些抗体均为市售。 </li><li>添加适当的人与生物圈计划（在预定的浓度）染色所需的细胞群，并培育20-30分钟冰在黑暗中，两个染色缓冲液洗。可选：一个重要的染料，如碘化丙啶染色，可以辨别死细胞（5）。如果是用于多色染色，单一颜色的控制是必需的。我们使用屋宇署CompBeads准备单一的颜色控制，使用制造商的协议。 </li><li>如果不使用直接标记的抗体，重复步骤7，使用适当的辅助抗体或链霉亲和素共轭。 </li><li>清洗后，重新悬浮培养液中的细胞，并确定使用至关重要的，如台盼蓝染料的细胞浓度。 </li><li>调整细胞浓度为15-20 × 10 6 / ml的。细胞群，形成集群，这可能会阻塞在排序仪器，通过一个过滤器过滤的细胞。 </li><li>设置和优化的细胞分选。设立流式细胞仪取决于制造过程是多种多样的，需要由受过适当训练的的人员执行。 一般建议如下： <ol style="list-style-type:lower-roman;"><li>取决于细胞类型进行排序，选择合适的喷嘴。 </li><li>对于无菌各种，消毒仪器。 </li><li>执行与珠激光器的运作，以验证仪器的质量控制，它是准确排序。 </li><li>安装所需的收集装置和侧流。 </li><li>看仪器的激光器和探测器成立，以确定可以使用（我们的BD流式细胞仪唱腔有三个激光头，可以检测多达九种颜色）的荧光标记。 </li><li>一旦确定将荧光标记的细胞分选使用，补偿可以执行（如下所述）。 </li></ol></li><li>阴性对照样品和单阳性对照进行补偿。补偿是必要的删除两个探测器之间的频谱重叠。这是有关赔偿是不是傻子证明是不利明亮的某些标记染色，从而导致暗淡和消极的人群之间的分辨率较差的低自体荧光的细胞，具有荧光的影响。 为了达到最佳效果，实验设计应包括使用明亮的荧光标记低表达或不具有已知的染色模式。标记之间的细胞群和高效表达提供良好的分离，应使用与二聚体的荧光，如红色或紫色激光器激发的。 <ol style="list-style-type:lower-roman;"><li>可以进行赔偿与补偿珠，这是聚苯乙烯微粒已耦合到一个特定免疫球蛋白κ轻链从鼠标，大鼠，大鼠/仓鼠的抗体的。珠子很容易染色，有一个强有力的信号，并准备作为实验样品具有相同的荧光团的单染控制提供了一个简单的方法。 </li><li>设置一个模板，其中包括一个二元图显示前向散射（FSC）和侧散射（SSC），和一个直方图每个将要使用的荧光。 </li><li>运行阴性对照管和调整前向散射（FSC）和侧向散射（SSC），以地方的人口规模的兴趣。 </li><li>调整荧光光电倍增管的设置，以地方人口负或直方图的左手部分的自体荧光。 </li><li>记录阴性对照管。 </li><li>运行单阳性对照管，并记录每个管数据。 </li><li>周围画直方图数据的积极部分，另一个直方图的负面部分的间隔门的时间间隔门。 </li><li>手动补偿是通过调整中位数（中位数是更好的集中趋势估计比平均对数刻度）的阳性，直到它等于底片的中位数。这项工作必须为每个实验中所用的荧光标签。 </li><li>要调整中位数，调整光谱重叠值或高或低，直到每个荧光参数匹配的中位数尽可能密切。 </li><li>补偿，也可以自动完成与分析软件。自动补偿使用补偿控制记录的数据来计算所有的实验中所用的荧光团的代数矩阵。使用这些值减去非主要的颜色是原色的探测器出血的贡献。 </li></ol></li><li>记录实验样品进行排序，并使用门控的工具和子集方法定义感兴趣的人群。 <ol style="list-style-type:lower-roman;"><li>用散点图显示FSC与SSC和使用门控工具，多边形，矩形，间隔或象限门的人口的利益，设置实验的模板。散点图显示，这是不同的细胞类型的细胞的物理性质，化学性质。 </li><li>确定荧光浇注战略的最佳方式是使用荧光减一（FMO）的控制。在鱼类统营处控制管实验中所用的所有试剂，包括利益的除外。鱼类统营处有助于区分依稀染色的人口和广阔的负面人口。 </li><li>鱼类统营处管使用的数据记录下来，以确定在何处放置实验管内的门。 </li></ol></li><li>一旦已经确定盖茨，盖茨可以选择到外部收集管的排序。最多四个门（或群体）可以在同一时间进行排序，取决于仪表。 </li><li>运行实验样品管4 ° C，打开偏转板，样品进行排序。 5 × 10 5 -1.5 × 10 6细胞可以分为12 × 75毫米的管道和1.5 × 10 6 - 4.5 × 10 6细胞可分为15 ml锥形排序。 </li><li>一旦已经得到所需数量的细胞，手动停止排序。 </li><li>执行后的排序分析，以确定排序的细胞群的纯度。 </li></ol>代表性的成果我们已经表明小鼠的脾B和CD4 T细胞排序（图1）的结果。脾细胞染色与体育得克萨斯红色标记的抗鼠B220，FITC标记的抗鼠TCRβ和Alexafluor - 700 -共轭抗小鼠CD4识别B细胞（B220 +TCRβ）和CD4 T细胞（CD4 +TCRβ+ ）。与BD流式细胞仪咏叹调仪器进行排序。排序后的B细胞纯度&gt; 97％的CD4 T细胞纯度&gt; 98％。 <img src="/files/ftp_upload/1546/1546fig1.jpg" alt="图1" /> 图1。纯度脾B细胞和CD4 T后，流式细胞仪的细胞 。小鼠脾细胞抗鼠B220，TCRβ和CD4抗体染色。 B细胞流式细胞仪上B220浇注使用BD流式细胞仪咏叹调+TCRβ细胞（图1A，左面板中，右下象限）。后的排序进行分析，以确定纯度的B细胞（图1A，右侧面板）。 B220染色显示在X轴和Y轴TCRβ染色。 CD4 T细胞的流式细胞仪进行浇注上使用相同的流式细胞仪TCRβ+ CD4 +细胞（图1B，左侧面板，右上象限）。后排序进行分析，以确定纯度的CD4 T细胞（图1B，右图）。 TCRβ染色上显示的X轴和Y轴染色的CD4。 ％的阳性细胞在每一个象限所示。 <!--<img src="/files/ftp_upload/1546/1546fig2.jpg" alt="图2" /> 图2。纯度脾脏CD4 T细胞后，流式细胞仪 ，小鼠脾脏T细胞富集去除B细胞通过平移。随后，细胞染色抗鼠TCRβ和CD4抗体（左侧面板）。流式细胞仪是使用BD流式细胞仪咏叹调​​TCRβ+ CD4 +细胞的门控（左侧面板，右上角quadr蚂蚁）。后的排序进行分析，以确定T细胞的纯度大于99％（右图）。 TCRβ染色上显示的X轴和Y轴染色的CD4。 ％的阳性细胞在每一个象限所示。 - &gt;

<ol><li>Herzenberg, L. A., De Rosa, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monoclonal+antibodies+and+the+FACS%3A+complementary+tools+for+immunobiology+and+medicine%5BTitle%5D)+AND+%22Immunol+Today%22%5BJournal%5D)">Monoclonal antibodies and the FACS: complementary tools for immunobiology and medicine.</a> Immunol Today. 21, 383-390 (2000).</li>
<li>Givan, A. L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGivan%2C+AL+%22Flow+cytometry+first+principles%22">Flow cytometry first principles</a>. , Wiley-Liss. New York. 273-273 (2001).</li>
<li>Hewitt, Z., Forsyth, N. R., Waterfall, M., Wojtacha, D., Thomson, A. J., McWhir, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence-activated+single+cell+sorting+of+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Cloning+Stem+Cells%22%5BJournal%5D)">Fluorescence-activated single cell sorting of human embryonic stem cells.</a> Cloning Stem Cells. 8, 225-234 (2006).</li>
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流式细胞仪是一个高度复杂的技术，为净化细胞群体的利益，可排序人口的纯度非常高（95-100％）。因此，这种技术是特别重要的实验，其中高纯度的一项基本要求（如微阵列分析）。流式细胞仪是其他可用的方法净化，特别是有利的，当一个细胞的人口需要每周表达的表面标志的基础上得到净化，或当两个或两个以上的人口有相同的表面标志表达的不同层次的需要得到净化。例如，净化边缘区B细胞（B220 + CD21 您好 CD23 INT /低 ）和滤泡B细胞（B220 + CD21 INT ​​/低 CD23 您好 ）根据CD21和CD23的表达水平。流式细胞仪的另一个增长的应用是单细胞分选，使单个细胞分析（3，4）。流式细胞仪是排序细胞表达荧光蛋白的基因表达，如绿色荧光蛋白标签蛋白（5），也常用来。如果执行无菌排序细胞可以培养的。然而，细胞活力和产量，可能会影响在排序。的可行性，可以改善通过使用排序细胞依赖的最佳温度和处理收集管后立即达到饱和。通过使用聚丙烯收集管，收集血清中丰富的媒体和反相收集管间歇的排序细胞，最佳的温度保持在收集管和离心〜10分钟（http://cyto.mednet.ucla.edu排序的细胞可以增加恢复/ Protocol.htm）。 超高速分拣机的发展扩大，在临床上的应用程序的流量排序的可能性。潜在的流式细胞仪的临床应用，包括达到治疗目的，在肿瘤治疗中的应用，取代羊膜穿刺术排序人类的精子和疾病早期检测（6，7），从人体血液中的造血干细胞的纯化。 多色染色，抗体选择是关键的一步，并为每个特定的仪器检测系统的基础上选择荧光。还有一些销售公司细胞分选机和激光配置各不相同，可根据用户需要。因此，人们不能简单地使用相同的抗体在发表的研究报告使用未经确认的兼容性。为了达到最佳的抗体选择的情况下，应考虑对被检查的蛋白质表达水平。在一般情况下，应该是最亮的可用荧光标记抗体染色隐约表达的表面标志物，可用于高表达的表面标志物（8）的染色，而暗淡的荧光。此外，抗体，应选择以这样的方式在他们的发射光谱重叠是最小的。多色染色，这是极其困难的选择荧光，没有光谱重叠。在这种情况下，执行赔偿。 补偿是必要的，以数学消除之间的排放，是由探测器（8-10）可测量各种荧光光谱荧光重叠。换句话说，是由荧光染料之一荧光重叠排放量的另一种荧光光谱减去。赔偿的计算需要一个不染控制每个荧光和单阳性对照，用于多色染色（9，10）。补偿可以进行细胞或珠子。珠子很容易染色，有一个强有力的信号，并准备作为实验样品有相同的荧光基团的单染控制提供了一个简单的方法。补偿珠时特别有利的表达和细胞表面蛋白的兴趣是在低水平的细胞群体的利益时是在初始的细胞群的限制。 一旦抗体已被选定，它们的活动应该优化执行滴定分析。如果流式细胞仪机具有非常强大的激光器，这一点尤为重要。最理想的染色抗体浓度的使用可能会导致分离所需的细胞群的细胞总人口的贫困。使用高浓度抗体，增加抗原的非特异性染色的机会。因此，抗体滴定将使抗体浓度的选择，给出了积极的人口和最小的背景染色（8）的最大亮度。 流式细胞仪现在是一个细胞亚群的净化标准技术。它可用于任何类型的细胞在单细胞悬液，可产生抗体，以确定所需的细胞群分开。流式细胞仪是首选的方法，当Highly纯的细胞群是必需的。

purification of specific cell population by fluorescence activated cell sorting (facs)

实验和临床研究，往往需要高度纯化的细胞群。流式细胞仪是一种选择的技术来净化称为表型的细胞群。净化等大宗方法包括平移，补充枯竭和磁珠分离。然而，流式细胞仪已超过其他可用的方法有几个优点。流式细胞仪是首选的方法时，需要非常高的纯度所需的人口，目标细胞群时，表示一个非常低的水平，识别标记或细胞群时，要求分离的基础上差标记密度。此外，流式细胞仪是唯一可用的净化技术来隔离内部染色或细胞内蛋白质的表达的细胞，如转基因荧光蛋白标记。流式细胞仪允许的大小，粒度和荧光的单个细胞的纯化。为了净化细胞的利益，他们是先染色与荧光标记的单克隆抗体（mAb），识别所需的细胞群（1）的特异性表面标志。未染色细胞的阴性选择也是可能的。流式细胞仪净化要求与排序能力和相应的软件的流式细胞仪。流式细胞仪，细胞悬浮液中通过包含在激光面前的每一个单细胞的液滴流。荧光检测系统检测细胞根据预定的细胞的荧光参数的利益。该仪器适用于一次充电到液滴含有的利益细胞和一个有利于收集到相应的收集管（2）带电液滴的静电偏转系统。染色，从而排序的成功在很大程度上取决于选择的识别标记和单克隆抗体的选择。排序参数，可以调整取决于纯度和产量的要求。虽然流式细胞仪需要专门的设备和人员培训，它是隔离的高度纯化的细胞群的首选方法。

Watch this Scientific Journal Video about Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS at JoVE.com

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

许多科学研究需要的细胞群的生物和化学分析，细胞必须在高纯度的状态。荧光激活细胞分选（FACS）是一种高级的方法，在其中获得纯的细胞群。

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

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Nanyang Technological University

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Immunology and Infection

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

73.2K 次观看.  BloodCenter of Wisconsin. 许多科学研究需要的细胞群的生物和化学分析，细胞必须在高纯度的状态。荧光激活细胞分选（FACS）是一种高级的方法，在其中获得纯的细胞群。

视频: Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

科研

生物学

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

生物工程

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

神经科学

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 ...

免疫与感染

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 of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

荧光活化细胞分选建立纯化的 Gabaep 或谷胱甘肽神经元的原代细胞培养

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

荧光激活细胞分拣，用于分离瘦杆菌细胞群

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

人类MK从人类主要来源的免疫细胞分拣或血细胞遗传基因 的体外 分化

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and ...

人间充质干细胞的荧光激活单细胞分选

Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and are known for their immunomodulatory and anti-inflammatory properties. The use of these stem cells is a boon to the field of regenerative biology, but at the same time, a bane to regenerative medicine and therapeutics owing to the multiple cellular ambiguities associated with them. These ambiguities may arise from the diversity in the source of these stem cells and from their in vitro growth conditions, both of which reflect upon their functional heterogeneity.
This warrants methodologies to provide purified, homogeneous populations of MSCs&#160;for therapeutic applications. Advances in the field of flow cytometry have enabled the detection of single-cell populations using a multiparametric approach. This protocol outlines a way to identify and purify&#160;stem cells from&#160;human exfoliated deciduous teeth (SHEDs) through fluorescence-assisted single-cell sorting. Simultaneous expression of surface markers, namely, CD90-fluorescein isothiocyanate (FITC), CD73-peridinin-chlorophyll-protein (PerCP-Cy5.5), CD105-allophycocyanin (APC), and CD44-V450, identified the &#34;bright,&#34; positive-expressors of MSCs&#160;using multiparametric&#160;flow cytometry. However, a significant drop was observed in percentages of quadruple expressors of these positive markers from passage 7 onwards to the later passages.
The immunophenotyped subpopulations were sorted using the single-cell sort mode where only two positive and one negative marker constituted the inclusion criteria. This methodology ensured the cell viability of the sorted populations and maintained cell proliferation post sorting. The downstream application for such sorting can be used to evaluate lineage-specific differentiation for the gated subpopulations. This approach can be applied to other single-cell systems to improve isolation conditions and for acquiring multiple cell surface marker information.

作者聚焦：单细胞分选在分离间充质干细胞纯化群体中的重要性 

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based approach.

人脱落乳牙免疫表型间充质干细胞的单细胞分选

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and ...

特定细胞群的净化荧光激活细胞分选（FACS）

摘要

探索更多视频

植物原生质体的荧光激活细胞分选

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

分离及小鼠淋巴细胞的激活

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

荧光活化细胞分选建立纯化的 Gabaep 或谷胱甘肽神经元的原代细胞培养

荧光激活细胞分拣，用于分离瘦杆菌细胞群

人类MK从人类主要来源的免疫细胞分拣或血细胞遗传基因的体外分化

人脱落乳牙免疫表型间充质干细胞的单细胞分选

特定细胞群的净化荧光激活细胞分选（FACS）

摘要

探索更多视频

植物原生质体的荧光激活细胞分选

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

分离及小鼠淋巴细胞的激活

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

荧光活化细胞分选建立纯化的 Gabaep 或谷胱甘肽神经元的原代细胞培养

荧光激活细胞分拣，用于分离瘦杆菌细胞群

人类MK从人类主要来源的免疫细胞分拣或血细胞遗传基因 的体外 分化

人脱落乳牙免疫表型间充质干细胞的单细胞分选

人类MK从人类主要来源的免疫细胞分拣或血细胞遗传基因的体外分化