这项工作是由美国国立卫生研究院拨款R01NS046835和R01AG034389，三月优生优育基金会授予6FY08 - 322的支持。特别感谢你致力于夫人埃莉诺布朗（美天旎生物技术，赤褐色，CA），极大地帮助她的洞察力到MACS科技。美天旎慷慨地提供实验示范使用，不需任何费用的材料。我也很感谢博士王广沪（医学院格鲁吉亚/格鲁吉亚卫生科学大学，奥古斯塔，佐治亚州）表达细胞株产生的PKCζ。也承认在格鲁吉亚/佐治亚州健康科学大学（下林美博士的董事）医学院分子医学研究所的支持。

1。简介在我们的实验室开发的脂质囊泡介导的亲和层析法，使用磁激活细胞分选（LIMACS）技术隔离酰胺相关的蛋白质复合物1-3。本来，脂质囊泡酰胺和磷脂酰丝氨酸，允许使用磁粒子标记的膜联蛋白V（高度仿射磷脂酰丝氨酸）隔离囊泡及其相关蛋白的Mac。我们已经使用的LIMACS 技术，在体外再造一个酰胺相关的极性复杂，从细胞神经酰胺结合蛋白的分离裂解液3。 LIMACS可以修改囊泡（例如，糖脂specifc凝集素或脂质抗体）的隔离使用其他互动的合作伙伴。 2。实验步骤脂质囊泡的制备和aPKCBinding分析<ol><li>脂质囊泡获得干混合物的摩尔量的磷脂酰丝氨酸（105微克）和C16 -神经酰胺（85微克）以下程序修改为大脂质体的制备1,4-7。 </li><li>脂质混合物，然后再悬浮和超声在100μL的50 mM的Tris /盐酸（pH值7.5）和150毫米氯化钠组成的囊泡缓冲区1小时。 </li><li>加入300辅以0.1毫米MnCl 2囊泡缓冲液后，样品离心20分钟12,000μ 克在4 ° C </li><li>颗粒（大脂质囊泡）是悬浮于100μL囊泡缓冲区，并在37℃1小时Vybrant CM - DII与1 nmol孵育° C至可视化磁珠分离后的囊泡分数。 Vybrant CM - DII是一种红色荧光染料，特别纳入到脂膜。 </li><li>洗涤剂无细胞裂解液，超声/ 300低渗缓冲离心去除膜碎片（10毫米的Tris / HCL（pH值7.0），蛋白酶和磷酸酶抑制剂）液中的细胞同质化的准备。应在1小时10 XG的离心步骤，以避免污染的细胞裂解液中含有内源性膜磷脂酰。 </li><li>清除裂解物被添加到脂质囊泡悬挂，混合物在4 ° C孵育2 h </li><li>反应混合物20μL20X Annexin V的结合缓冲液和50μL含有磁珠标记Annexin V的解决方案的补充，其次是潜伏期为1小时在4 ° C </li><li> MACS是根据制造商的协议（美天旎生物技术公司）。脂质囊泡的存在和数量是由监察Vybrant CM - DII通过流量和洗脱用酶标仪荧光读者分数​​荧光。 </li><li>水泡脂量和荧光强度之间的线性相关囊泡约束Vybrant CM - DII是由高性能定量薄层色谱法（薄层）应用于膜联蛋白V的Mac脂混合物验证。 </li><li> aPKC或其他蛋白质的结合反应的特异性神经酰胺/磷脂酰丝氨酸的囊泡竞争，使用1微克的反PKCζ兔多克隆抗体孵育1 h后的细胞裂解液在4 ° C到孵化前的抗体检测验证脂质囊泡。 </li><li> SDS - PAGE电泳和免疫印迹分析蛋白结合的Mac洗脱液酰胺/磷脂酰丝氨酸的囊泡。 </li></ol> 在体外脂质蛋白的极性复杂<ol><li> 在体外重组脂质蛋白极性复杂LIMACS程序后，在上一节所述。在简短，磷脂酰丝氨酸（420微克）和C16 -神经酰胺（107微克）是有机溶剂干燥。 </li><li>干血脂悬浮下超声在500囊泡缓冲液（50 mM的三/盐酸，pH值7.5; 150 mM氯化钠）。 </li><li>五μL10 mM的氯化锰，1μLVybrant CM - DII，和500PKCζ吴（重组人）和反应混合物孵育60分钟undergentle鼓动在4 ° C。 </li><li> 60分钟在4 ° C离心Vybrant CM - DII染色的磷脂酰丝氨酸/酰胺囊泡回收12000 XG </li><li>颗粒（粉红色），悬浮于100μLTris缓冲液，并辅以γS- GTP（100μM），国内生产总值（1毫米），GST - Par6（100毫微克），或（500纳克）GST - Cdc42的，并进一步培养3在4 h ° C（ 任何其他利益的重组蛋白结合，这里可以使用） 。 </li><li> Annexin V的缓冲液（5μL20倍原液）和Annexin V的共轭磁珠（50μL）的增加和反应混合物轻轻摇动下孵育另一个30分钟，4 ° C。 </li><li>膜联蛋白V - MACS执行以下供应商的协议，如前所述。 </li&gt; <li>洗脱分数（1毫升）补充10微克的纯卵清蛋白沉淀援助。韦塞尔- Flugge降水集中的蛋白质和SDS-PAGE/immunoblotting如先前所描述的8分析。 </li><li>量化Vybrant CM - DII（粉红色）在有机相（氯仿/甲醇）韦塞尔Flugge沉淀反应检测洗脱脂质囊泡的数量。分析蛋白质的量是等量的脂质囊泡归。 </li></ol> 3。结果 LIMACSPKCζ- EGFP和神经酰胺结合域C20ζ- EGFP的净化一个洗涤剂无裂解MDCK细胞表达全长PKCζC端与绿色荧光蛋白（FLζEGFP）基因或神经酰胺在PKCζ（C20ζ- EGFP）C -末端结合域的培养与磷脂酰丝氨酸/酰胺囊泡描述了实验步骤。 MACS柱洗脱后，用免疫印迹和PKCζ和EGFP的检测洗脱蛋白2抗体蛋白进行了分析。 <img src="/files/ftp_upload/2657/2657fig1.jpg" alt="图1" /> 图1。LIMACS EGFP -标记PKCζ和C -末端片段C20ζ使用磷脂酰丝氨酸/酰胺囊泡。 MDCK细胞表达EGFP（作为一个不具约束力的的控制），全长PKCζ- EGFP，或神经酰胺结合，C -末端片段C20ζ- EGFP的洗涤剂裂解液孵育实验程序部分所述与磷脂酰丝氨酸/酰胺囊泡。使用LIMACS后，SDS样品缓冲液洗脱蛋白，并用SDS - PAGE分析和免疫印迹。左侧面板显示，EGFP的没有约束力的V -膜联蛋白磁珠保留水疱。中间和右边的面板显示，全长PKCζ- EGFP和C20ζ- EGFP的保留绑定酰胺。

<ol>
	<li>Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+binding+to+ceramide+activates+protein+kinase+Czeta+before+the+formation+of+a+pro-apoptotic+complex+with+PAR-4+in+differentiating+stem+cells.">Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells.</a> J Biol Chem. 280, 26415-26424 (2005).</li><li>Wang, G., Krishnamurthy, K., Umapathy, N. S., Verin, A. D., Bieberich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+carboxyl-terminal+domain+of+atypical+protein+kinase+Czeta+binds+to+ceramide+and+regulates+junction+formation+in+epithelial+cells.">The carboxyl-terminal domain of atypical protein kinase Czeta binds to ceramide and regulates junction formation in epithelial cells.</a> J Biol Chem. 284, 14469-14475 (2008).</li><li>Chalfant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+ceramide+regulates+the+alternative+splicing+of+caspase+9+and+Bcl-x+in+A549+lung+adenocarcinoma+cells.+Dependence+on+protein+phosphatase-1.">De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.</a> J Biol Chem. 277, 12587-12595 (2002).</li><li>Simon, C. G., Holloway, P. W., Gear, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exchange+of+C(16)-ceramide+between+phospholipid+vesicles.">Exchange of C(16)-ceramide between phospholipid vesicles.</a> Biochemistry. 38, 14676-14682 (1999).</li><li>Goni, F. M., Contreras, F. X., Montes, L. R., Sot, J., Alonso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysics+(and+sociology)+of+ceramides.">Biophysics (and sociology) of ceramides.</a> Biochem Soc Symp. , 177-188 (2005).</li><li>Kumagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CERT+mediates+intermembrane+transfer+of+various+molecular+species+of+ceramides.">CERT mediates intermembrane transfer of various molecular species of ceramides.</a> J Biol Chem. 280, 6488-6495 (2005).</li><li>Wessel, D., Flugge, U. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+quantitative+recovery+of+protein+in+dilute+solution+in+the+presence+of+detergents+and+lipids.">A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.</a> Anal Biochem. 138, 141-143 (1984).</li></ol>

要测试的脂质和蛋白之间的相互作用，是阻碍嵌入细胞膜的脂质。细胞膜由一些脂类和蛋白质的混合​​物，它是在组织脂质微区或木筏。因此，微区和蛋白质的合作，净化不能明确区分，如果一种蛋白质直接结合到脂质，或仅仅是在微区结构丰富。使用涂层表面上，如脂质酶联免疫吸附或等离子体共振光谱的定义血脂的其他方法可以检测其结合蛋白的一个特定的脂质相互作用..然而，在一个有?...

Annexin V的共轭磁珠和Macs和微观minicolumns提供了由美天旎生物技术公司（赤褐色，CA）。血脂最高纯度和阿凡提极性脂（雪花石膏，AL）获得。

lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (limacs): a novel method to analyze protein-lipid interaction

脂蛋白质相互作用的分析是困难的，因为嵌在细胞膜的脂质，因此，无法以最净化程序。血脂作为一种替代方法，可以在平坦的表面涂层，为脂质ELISA法和等离子体共振光谱。然而，表面涂层血脂不形成微区结构，这可能是重要的脂质绑定属性。此外，这些方法不容许较大数额的蛋白结合他们的目标血脂净化。 为了克服这些限制测试脂蛋白质相互作用和净化脂质结合蛋白，我们开发了一种新的方法称为脂质囊泡介导的亲和层析法，使用磁激活细胞分选（LIMACS）。在此方法中，脂质囊泡锚脂质膜联蛋白V的Mac与目标脂质和磷脂酰准备。磷脂是一个无处不在的细胞膜磷脂，高亲和力的蛋白膜联蛋白V。使用Annexin V的共轭含有的磷脂脂质囊泡结合磁珠磁珠。当脂质囊泡与细胞培养裂解的蛋白结合的目标脂质也将受到约束珠，可以共同使用Mac纯化。这种方法也可以用来测试一个目标脂质蛋白复合物结合，如果重组蛋白重组。 我们已经用这种方法，以显示与鞘脂酰胺和合作净化前列腺凋亡反应4（4杆），蛋白结合酰胺相关aPKC非典型蛋白激酶C（aPKC）的相互作用。我们也使用这种方法，重组的重组aPKC神经酰胺与细胞极性相关的蛋白质Par6和Cdc42复杂。由于脂质囊泡可以与各种sphingo或磷脂准备，LIMACS脂质 - 蛋白相互作用密切类似于细胞膜的脂质环境提供了通用的测试。其他脂质蛋白复合物可以识别使用的脂质结合蛋白与脂质囊泡的合作纯化的蛋白质组学分析。

Watch this Scientific Journal Video about Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction at JoVE.com

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

为了测试我们使用Macs和Annexin V的共轭磁珠和目标脂质和Annexin V的具有约束力的磷脂酰丝氨酸合成脂质囊泡与目标脂质的一种蛋白质的相互作用。绑定到目标脂质蛋白质纯化和分析后，从珠洗脱。

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）：一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

lipid-vesicle-mediated-affinity-chromatography-using-magnetic

Research

JoVE Journal

Biology

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

12.4K 次观看.  Georgia Health Sciences University. 为了测试我们使用Macs和Annexin V的共轭磁珠和目标脂质和Annexin V的具有约束力的磷脂酰丝氨酸合成脂质囊泡与目标脂质的一种蛋白质的相互作用。绑定到目标脂质蛋白质纯化和分析后，从珠洗脱。

视频: Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

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 Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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 ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.

结合母细胞和波动测试磁分选，以分析基因组不稳定性的过程中有丝分裂细胞衰老<em&gt;酿酒酵母</em

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

本机配合物的净化对结构研究使用串联亲和标签法

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

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 ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...