从夫人祈福长老白格雷厄姆的赠款支持这项工作是由资助研究基金和美国国立卫生研究院（AR059838）CB，由美国国立卫生研究院（AR052791，AR044387复苏法案，NS063298）LTT和贝勒医学院医学流式细胞仪和细胞分选的资金来自美国国立卫生研究院（NCRR S10RR024574，NIAID的AI036211，美国国立癌症研究所P30CA125123）的核心。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

1。细胞染色<ol><li>准备细胞。同时，仍在积极成长（60 - 80％汇合），应采用细胞。血清的细胞饿死在一夜之间，在0.5毫升血清在24孔板和孵化器的媒介，每口井0.1万盘他们的细胞（37℃，5％的CO 2）为30分钟- 2小时恢复从胰蛋白酶。 </li><li>混合小学和中学的抗体。混合（5μL的主要反GLUT4的抗体与1μL，中学鸡抗山羊IgG抗体结合AlexaFluor 488）x水井数。孵育10分钟在室温下，在黑暗中。 </li><li>准备培养基中稀释的胰岛素2X工作股票。 </li><li>检查，如果细胞已加入或不。 </li><li>轻轻加入6μL抗体混合胰岛素2X工作股票和0.5毫升含有细胞的井。避免任何介质的动荡。孵育（37 ° C，5％的CO 2）为30分钟，在黑暗中。 </li><li>每孔加入0.5 mL的PBS + 1％PFA无晃动的细胞，修复细胞。孵育20分钟在室温下，在黑暗中。 </li><li>如果你的细胞一直坚持到井，轻轻抬起一个细胞起苗。转移到适合您的流式细胞仪，离心沉淀细胞管细胞（共1毫升）。 </li><li>洗为0.4 mL PBS用1 mL PBS和重新悬浮颗粒的两倍+ 1％的煤灰。此时，细胞可在铝箔包裹，并储存在冰箱中（4-8 ° C），或采取即时数据采集的流式细胞仪。 </li></ol> 2。数据采集<ol><li>设置一个广泛的门，围绕在侧散射（SSC）与前向散射（FCS）的面板的活细胞。使用负管（不与胰岛素或细胞"染色"的同型对照治疗的细胞），设置FL1参数。确保您的高峰期是在面板上，而不是完全切下侧。 </li><li>至少10000个管细胞获取数据。 </li></ol> 3。数据分析<ol><li>在SSC和FSC面板（图1A）活细胞周围绘制一个门。 AlexaFluor 488荧光强度与的活细胞门内的细胞（图1B）数绘制的直方图。您可以覆盖不同的直方图，直观的差异，但定量分析，确定每个单元的人口的平均数和中位数的荧光强度，并使用这些数字，你的比较（图1C）。 </li><li>规范化数据在胰岛素的情况下得到的数值，在图1C和1D所示。 </li></ol> 4。代表性的成果从一个健康的人（无胰岛素抵抗）中分离出的成肌细胞，胰岛素诱导的GLUT4从质膜的细胞质中（图1，左）的剂量依赖性易位。与此相反，胰岛素不诱导GLUT4转位与胰岛素抵抗的患者（图1，右）从孤立的成肌细胞的细胞膜。 <img src="/files/ftp_upload/2429/2429fig1.jpg" alt="图1" /> 图1。染色实验的例子。成肌细胞是从捐助者无胰岛素抵抗（左）与胰岛素抵抗的患者（右）从孤立的，SSC（侧散射）与FCS的（前向散射）情节与门周围的活细胞，B细胞，门控一，对外部抗原表位的GLUT4的抗体染色，并留下未刺激（红色）或刺激胰岛素（蓝色）1纳米，10纳米（橙色），或100纳米（绿色）。对于图的清晰度，我们只显示数据没有与100纳米胰岛素与胰岛素抵抗的细胞 C，平均荧光强度（MFI）在B所示的直方图规范化左未刺激细胞的MFI（无胰岛素） 。 研发 ，小额信贷机构的规范化直方图数据的图形显示在B

<ol>
	<li>Zhao, F. Q., Keating, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+and+genomics+of+glucose+transporters.">Functional properties and genomics of glucose transporters.</a> Curr. Genomics. 8, 113-128 (2007).</li><li>Karnieli, E., Armoni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+the+insulin-responsive+glucose+transporter+GLUT4+gene:+from+physiology+to+pathology.">Transcriptional regulation of the insulin-responsive glucose transporter GLUT4 gene: from physiology to pathology.</a> Am. J. Endocrinol. Metab. 295, 38-45 (2008).</li><li>Graham, T. E., Kahn, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+alterations+of+glucose+transport+and+molecular+mechanisms+of+intertissue+communication+in+obesity+and+type+2+diabetes.">Tissue-specific alterations of glucose transport and molecular mechanisms of intertissue communication in obesity and type 2 diabetes.</a> Horm. Metab. Res. 39, 717-721 (2007).</li><li>Alter, G., Malenfant, J. M., Altfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD107a+as+a+functional+marker+for+the+identification+of+natural+killer+cell+activity.">CD107a as a functional marker for the identification of natural killer cell activity.</a> J. Immunol. Methods. 294, 15-22 (2004).</li><li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutellingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labeled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V.</a> J. Immunol. Methods. 184, 39-51 (1995).</li><li>Lugli, E., Roederer, M., Cossarizza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+analysis+in+flow+cytometry:+the+future+just+started.">Data analysis in flow cytometry: the future just started.</a> Cytometry A. 77, 705-713 (2010).</li></ol>

我们已经证明了量化从细胞质流式细胞仪检测细胞质膜的GLUT4转位的快速技术。 GLUT4的是瞬时表达细胞的细胞膜内吞。由于仍然连接到GLUT4的内吞作用，即使在绑定的抗体，荧光信号给总额的GLUT4在细胞膜暴露在胰岛素存在的时间选择在孵化。 这种技术也可以应用于其他蛋白质的研究，从一个细胞内舱质膜的易位，提供了一个很好的向外部利益的蛋白质抗原表位的抗体。例如，一个类似的实验是现在常用的评估细胞毒性T淋巴细胞和自然杀伤细胞脱颗粒率，测量他们的质膜4,5 CD107a易位。磷脂酰丝氨酸从质膜的外侧在细胞凋亡或坏死的内侧易位还可以通过流式细胞仪检测，采用荧光标记的Annexin V的6。 除了速度的测定，流式细胞仪提出了允许在混合细胞群的质膜蛋白质易位研究的优势。事实上，我们可以结合染色细胞亚群的标记和感兴趣的蛋白质，多激光流式细胞仪数据采集。 该协议第1.2段中抗体的金额起点，我们建议你与你的细胞滴定的抗体，以确定最低的抗体浓度，为您提供了最大的信号。一个很好的起点主抗人GLUT4的抗体，我们这里使用的范围，将1-10μL每管（即0.2-2微克每管抗体），每管含有不超过1亿个细胞。对于其他的主要抗体，是指供应商的指示。 细胞自体荧光会有所不同从捐助者，捐助者和实验，实验数据的规范化是必要的的。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Anti-human GLUT4 antibodies</td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnologies</td>
<td>sc-1606</td>
<td>Against an external epitope of GLUT4</td>
</tr>
<tr>
<td>Chicken anti-goat IgG conjugated to AlexaFluor 488</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-21467</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human insulin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I2643</td>
<td>&nbsp;</td>
<tr>
<td>Cell lifters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>29942-118</td>
<td>Cut to fit the wells</td>
</tr>
<tr>
<td>5 ml tubes for FACS</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>60819-138</td>
<td>Fit all BD machines</td>
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quantitative measurement of glut4 translocation to the plasma membrane by flow cytometry

葡萄糖是身体能量的主要来源，因此需要不断调节其血药浓度。胰岛素由胰腺释放诱导胰岛素敏感的组织中，最引人注目的是大脑，骨骼肌和脂肪细胞对葡萄糖的摄取。患有2型糖尿病和/或肥胖的患者往往胰岛素抵抗和无法控制自己的血糖平衡。进入新的见解胰岛素抵抗的机制，可能为2型糖尿病提供新的治疗策略。 过剩的葡萄糖转运家族由十三名成员，分布在不同的组织遍布全身 1 。胰岛素敏感组织，如骨骼肌介导的葡萄糖摄取葡萄糖转运子4（GLUT4）是主要的转运。胰岛素与其受体的结合后，囊泡含有GLUT4的易位从细胞质到质膜，诱导葡萄糖的摄取。降低GLUT4转位是在2型糖尿病2,3中的胰岛素抵抗的原因之一。 从细胞质到质膜的GLUT4转位可通过免疫组织化学显示，使用荧光标记的GLUT4的特异性抗体。 在这里，我们描述了一个量化的GLUT4转位的总金额在选定的时间细胞质膜的技术，用流式细胞仪检测。此协议是快速（不少于4小时，包括孵化与胰岛素），并允许少3000细胞，或在一次实验中多达1万个条件细胞的分析。它依赖于外部抗原表位的转运反GLUT4的抗体绑定到它，因为它是暴露后易位到质膜的细胞外介质尽快。

Watch this Scientific Journal Video about Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry at JoVE.com

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

该协议描述了一种快速的技术量化从细胞质GLUT4转位的流式细胞仪检测细胞质膜。

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

quantitative-measurement-glut4-translocation-to-plasma-membrane-flow

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

26.4K 次观看.  Baylor College of Medicine. 该协议描述了一种快速的技术量化从细胞质GLUT4转位的流式细胞仪检测细胞质膜。

视频: Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

科研

生物学

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM:免疫沉淀，流式细胞仪检测

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

测绘在细胞膜分子扩散，多目标跟踪（MTT）

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

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 ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

视黄醇运输的血浆视黄醇结合蛋白的膜受体的实时分析

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

pH 依赖性荧光 Keima 对流式细胞术 Mitophagy 的敏感性测定

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.