我们感谢他们的帮助，流式细胞仪分选博士Yitzchak Oschry和博士ORIT的鹭阿西夫。这项研究得到了补助NE从欧盟第七框架计划（FP7/2007-2013）根据赠款协议Ň°[276890]，从以色列癌症协会（20110078），并从以色列癌症研究基金（ 研究职业发展奖 ）。

1。解剖小鼠乳腺皮肤组织<ol><li>解剖组织的小鼠之前，需准备以下用品及试剂: 耗材: <ul><li>外科工具（在用洗涤剂和洗涤，然后浸渍在70％的乙醇）。 </li><li>鼠标停留在剥离时的发泡胶面。 </li><li>引脚或针头在解剖小鼠。 </li><li>玻璃瓶盖的消化，含有磁性搅拌棒（灭菌）。 </li><li>水浴中于37℃，含有潜式磁力搅拌器和足够的水来淹没消化罐，休息时的搅拌盘上。 </li></ul>试剂: <ul><li> FACS缓冲I:（4°C）的Dulbecco磷酸盐缓冲液钙，镁，CMF（免费）+ 0.5％BSA </li><li>胶原酶溶液（使用前）: 0.05克Ⅱ型胶原酶 0.05克Ⅳ型胶原酶 0.01克Deoxyribonuc租赁 20毫升FACS缓冲液I.查看胶原酶溶液在冰上直至使用。 </li><li> PharmLyse（氯化铵溶胞试剂）。 </li><li> FACS缓冲液II:Dulbecco氏磷酸盐缓冲盐水CMF + 1％FCS的。 </li></ul></li><li> 乳腺解剖 （见17）。 <ul><li>安乐死使用二氧化碳（CO 2）的吸入的雌性小鼠。 </li><li>在一个塑料表面，将鼠标放在它的背部和脚牢牢四肢，用25号针（ 图1A）。鼠标慷慨喷用70％的乙醇。 </li><li>使用产钳，拉腹部皮肤在中线作一小切口，用锋利的剪刀。 </li><li>从开始的切口，切开皮肤动物的脖子上，同时避免对腹腔或胸腔穿刺。 </li><li>从正中切口朝向腿部上的后脚切开皮肤，导致在一个"Y形"。 </li><li>拉皮肤距离谅解备忘录Ê身体和牵制，露出附着到皮肤的底面（ 图1B）的乳腺。 </li><li>用棉签轻轻分开乳腺皮肤，同时降低结膜组织分开乳腺小锋利的剪刀对脊柱的鼠标。 </li><li>腹部的腺体（＃4，5）本协议是最好的。您还可以使用第二届乳腺，但请记住，这些腺体的解剖学位于靠近胸大肌这可能是很难区分，导致在混合组织起源的细胞。 </li><li>注:解剖肿瘤组织时，建议切除坏死的地区。 </li></ul></li><li> 皮肤组织的解剖:皮肤组织的最简单的方式来获得，而无需处理脱毛是使用鼠标的耳朵。如果大量的组织是必需的，你可以剃去头发从鼠标躯干和剖析皮肤。 <ul><li>安乐死的小鼠ü唱CO 2吸入。 </li><li>使用锋利的剪刀，立即切断的耳朵和地方在消化罐的体积小（约0.5毫升）的PBS冰。 </li></ul></li></ol> 2。的乳腺/护肤单细胞悬液的制备<ul><li>将乳腺/皮肤消化jar文件含有一个小体积的PBS在冰上。如果组织是"血腥与PBS洗X2"，然后丢弃多余的PBS。 </li><li>切碎，彻底弯曲的剪刀。 </li><li>加入20 mL胶原酶溶液（注:这将有效量的胶原酶溶液分离约5克的乳腺肿瘤组织和耳朵了15只）。 </li><li>将立即，搅拌板在37°C水浴，并孵育15分钟，而在中速搅拌。 </li></ul> 注:孵育时间可能有所不同，如果消化其他组织。 <ul><li>要停止反应，加入30毫升冷DMEM + 10％FCS。 </li><li>应变的组织/媒体混合物通过一个70微米的细胞过滤网放置在一个50毫升锥形管的顶部。 </li><li>离心锥形管中在450×g离心5分钟，在4℃下</li></ul> 3。红血细胞裂解 注意:这一步是没有必要，如果小鼠心脏灌注PBS处死。 <ul><li>吸取上清，使用的玻璃移液管连接到真空中，而不会干扰细胞沉淀。 </li><li>以溶解红细胞，细胞沉淀重悬在10毫升PharmLyse溶液并在室温下孵育5分钟。 </li><li>中和该PharmLyse溶液，加入约40毫升的FACS缓冲液一</li><li>附带的锥形管中，细胞在450×g离心5分钟，在4℃下</li><li>吸取上清液</li></ul> 4。阻断内源性的Fc <ul><li>可选:计数细胞，以确定适当的音量以下STEPS。 </li><li>悬浮细胞在1-3毫升FACS缓冲I（根据细胞数量及金额您要使用的抗体）。 </li><li>添加FcBlock（抗小鼠CD16/CD32）达到1:50稀释（10微克/毫升），并在冰上孵育10-20&#39;。 不需要洗掉FcBlock。 </li></ul> 5。染色法<ul><li>取出100μl等分试样被用来作为一种未染色的控制和单一的颜色控制（根据使用的荧光抗体的数目）的eppendorf管中。 </li><li>标记成纤维细胞:抗PDGFRα（直接结合的荧光）为1:50的稀释（10微克/毫升）。 </li><li>至标记其他细胞群（ 例如，免疫细胞，巨噬细胞，内皮细胞等）:添加适当的荧光共轭的表面标志物的抗体，以1:50稀释。 </li></ul> 注意:虽然PDGFRα是一个健壮的成纤维细胞标记物，它建议包括抗体对于immune细胞和上皮细胞，以排除任何双阳性的人群，从而提高了孤立的成纤维细胞的纯度。 <ul><li>添加2微升每种抗体的每个的单一颜色控制eppendorf管中。 </li><li> 1小时上冰在黑暗中培养细胞的抗体。 </li><li>洗:填写管​​FACS缓冲液I和旋转450×g离心5分钟，在4°C。 </li><li>吸液上清，重悬细胞在FACS缓冲I至浓度约为2×10 6个细胞/毫升，或两者浓度最适当的排序与您的可用FACS分拣机。 </li></ul>可选:细胞可以悬浮在FACS缓冲液II中的持续时间排序。这可能会提高细胞活力。然而，暴露于血清中的因素可能影响基因的表达，这应该被测试，并考虑。 <ul><li>标记的细胞转移到FACS管与管的过滤器:过滤单元防止细胞团块。 </li><li>排除死细胞，加入7AAD（0.5微克/毫升）或碘化丙啶（PI）的样品（未染色的控制除外）。 </li></ul>注意:为了避免浪费细胞，你可以添加7AAD/PI录制后，在流式细胞仪的未染色的样品，并且再次使用控制7AAD只。 <ul><li>在分析样本保持在冰上。 </li></ul> 6。通过流式细胞仪分离细胞 （注:该协议的一部分，需要经营FACS分选机， 如 BD FACS AriaII，或熟练的技术人员的协助下，在以前的知识）。 <ul><li>使用流式细胞仪分选机软件（BD FACSDiva），准备适当的分析图，分析前向散射（FSC），侧向散射光（SSC），7AAD，FITC，PE和任何其他荧光标记细胞（ 图2）。 </li><li>在加载每个样品的细胞分选仪，涡流简单地重悬细胞。 </li><li>首先分析仪的ING未染色样品以便设置总细胞群体的选通，栅极出细胞碎片，并以确定的自发荧光或背景染色。 </li><li>染色的样品+ 7AAD分析，以确定和门活细胞与细胞总人口的人口。 </li><li>分析每一个单一的颜色控制，补偿等，以确定和校准参数。 </li><li>染色的样品分析，定义的门的位置进行排序。 </li><li>一旦参数和门已设置为所需的细胞群，开始排序到微量离心管或15毫升锥形管中含有培养基或RNA裂解液（如RLT缓冲或Trizol法）标记的细胞群。 </li></ul>注意:如果大量的单元格进行排序，它是不建议直接进入分类，RNA裂解缓冲液，作为鞘液的大量伴随细胞的裂解缓冲液稀释，并降低产量。 <ul><li>灵宝n细胞后马上分拣完成后，转移到新鲜培养基或RNA裂解缓冲液，根据你的实验的目的。 </li><li>如果排序，以培养分离出的细胞，进行无菌排序。为了更好地恢复细胞，用无酚红DMEM + 5％FCS的FACS缓冲液II和20％FCS收集细胞培养液中。 </li><li>如果排序培养成纤维细胞，胶原蛋白涂层板alwaysplate，从来没有直接对塑料激活的成纤维细胞基因表达的变化，因为这结果。 </li></ul>

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没有利益冲突的声明。

虽然在组织培养中进行的实验可以是信息性 ​​的和建议可以验证在体内的功能原理，它是已知的，会发生大的变化，在基因表达的细胞在培养11,12。为了避免组织培养一步成纤维细胞中的基因表达分析时，我们开发了一个协议，允许隔离正常，以及与癌症相关的新鲜小鼠成纤维细胞或人体组织。该协议已成功应用于鼠标，从9人的皮肤，胰腺，乳腺组织。用流式细胞仪分?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;的试剂名称</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品目录号</strong</td&gt;<td<strong评论（可选）</strong</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM</td&gt;<td&gt; Gibco公司</td&gt;<td&gt; 41965</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PBS</td&gt;<td&gt;生物产业</td&gt;<td&gt; 02-023-1A</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;Ⅱ型胶原酶</td&gt;<td&gt;沃辛顿</td&gt;<td&gt; LS4176</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;Ⅳ型胶原酶</td&gt;<td&gt;沃辛顿</td&gt;<td&gt; LS4188</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;脱氧核糖核酸</td&gt;<td&gt;沃辛顿</td&gt;<td&gt; LS2007</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PharmLyse</td&gt;<td&gt; BD</td&gt;<td&gt; 555899</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;细胞过滤70微米</td&gt;<td&gt; SPL</td&gt;<td&gt; 93070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td纯化的抗小鼠CD16/CD32</td&gt;<td&gt; BD Pharmingen公司</td&gt;<td&gt; 553142</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;通过探测（7AAD）</td&gt;<td&gt; E-生物科学</td&gt;<td&gt; 00-6993-50</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; ANTI-MOUSE的CD140a - PE（PDGFRA）</td&gt;<td&gt; E-生物科学</td&gt;<td&gt; 12-1401-81</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;抗鼠F4/80- FITC</td&gt;<td&gt; Cederlane</td&gt;<td&gt; CL8940F</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM W / O酚红</td&gt;<td&gt; Gibco公司</td&gt;<td&gt; 31053</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; I型胶原</td&gt;<td&gt; BD Biosciences公司</td&gt;<td&gt; 354236</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

isolation of normal and cancer-associated fibroblasts from fresh tissues by fluorescence activated cell sorting (facs)

癌症相关的成纤维细胞（CAFS）是最重要的细胞类型，许多癌症的肿瘤间质内，特别是乳腺癌，其突出的存在往往与预后不良1,2。 CAF的活化的基质成纤维细胞的亚群，其中许多表达的肌纤维母细胞标记物α-SMA的3。 CAF的起源来自局部组织的成纤维细胞，以及从招募到显影的肿瘤和采用的CAF表型的影响下的肿瘤微环境4的骨髓来源的细胞。 CAF的信号，促进肿瘤细胞增殖，血管生成和侵袭5-8，促进肿瘤的发生，生长和发展。我们表明，CAF的提高肿瘤的生长，介导肿瘤促进炎症开始，最早在癌前阶段9。尽管越来越多的证据中发挥的关键作用CAF的促进肿瘤的生长，研究CAF的一直是一个持续的挑战，由于缺乏CAF-这些细胞的特异性标志物和广阔的异质性，与现有的许多亚型肿瘤微环境的10。此外，成纤维细胞的体外研究是通过阻碍它们的基因表达中的事实，常常在组织培养11,12改变。为了解决这个问题，让公正的新鲜小鼠的成纤维细胞和人体组织的基因表达分析，荧光激活细胞分选（FACS）13,14以前的协议的基础上，我们开发了一种方法。我们的方法依赖于利用PDGFRα隔离成纤维细胞的表面标记，从新鲜小鼠和人体组织。 PDGFRα中大量表达的正常成纤维细胞和纤维母9,15。此方法允许隔离纯种群的正常成纤维细胞和CAF的，包括，但不限于α-SMA +活化肌成纤维细胞。然后可以隔离成纤维细胞用于表征和比较基因的表达，在肿瘤发生在纤维母的演变。事实上，我们和其他人表达谱成纤维细胞分离细胞分选16。该协议成功地进行了高度浓缩的人群皮肤，乳腺，胰腺和肺组织中的成纤维细胞进行隔离和剖析。此外，我们的方法允许排序的细胞培养，在为了执行功能的实验，以避免污染的肿瘤细胞，这往往是一个很大的障碍时，试图来培养的CAF。

使用PDGFRα隔离高度富集的组织的成纤维细胞的种群中的成纤维细胞的结果作为一个标记。排序后的纯度为99％的水平，量化排序后分析（ 图2A）。估算污染的非成纤维细胞通过小区固有控制基因的相对表达量（ 图2B）的百分比通常显示0.1-0.6％的污染。这个级别的纯度，使高品质的转录组分析孤立的成纤维细胞9。 在乳腺中，在组织中的成纤维...

Watch this Scientific Journal Video about Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS at JoVE.com

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

癌相关成纤维细胞（CAFS）信号，促进细胞增殖，血管生成和炎症促进肿瘤的发生，生长和发展。在这里，我们描述了一种方法，分离出纯净的新鲜小鼠和人体组织的正常成纤维细胞和纤维母细胞分选，表面标志使用PDGFRα的人口。

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 ...

isolation-normal-cancer-associated-fibroblasts-from-fresh-tissues

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

35.6K 次观看.  Tel Aviv University. 癌相关成纤维细胞（CAFS）信号，促进细胞增殖，血管生成和炎症促进肿瘤的发生，生长和发展。在这里，我们描述了一种方法，分离出纯净的新鲜小鼠和人体组织的正常成纤维细胞和纤维母细胞分选，表面标志使用PDGFRα的人口。

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

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

科研

生物学

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 ...

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

溴脱氧尿嘧啶核苷（BrdU）标记和文化独立的鉴定溶解有机随后的荧光激活细胞分选碳有辱人格的浮游细菌

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

大鼠门户成纤维细胞的分离<em&gt;原位</em&gt;肝脏灌注

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

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 ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

单细胞微吸气作为荧光激活细胞分类的替代策略，用于巨型病毒混合物分离

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

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 ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

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）

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分离纯化果蝇外周神经元