この作品は、ミセスクリフォードエルダーホワイトグラハムがCBに、NIH（AR052791、AR044387 - ARRA、＆NS063298）からLTTに研究基金とNIHの（AR059838）寄附からの補助金によって、医学サイトメトリーのベイラー大学によってサポートされていましたNIH（NCRR S10RR024574、NIAID AI036211、＆NCI P30CA125123）からの資金でコアをソートしてセル。内容はもっぱら著者の責任であり、必ずしもNIHの公式見解を表すものではありません。

1。細胞の染色<ol><li>細胞を準備します。それでも積極的に（ - 80％コンフルエント60）成長中の細胞を使用する必要があります。回復するために2時間-血清は、一晩30分間インキュベーター内で24ウェルプレートと場所に0.5 mLの無血清培地（37℃、5％CO 2）にも当たり0.1万でプレートそれらの細胞を細胞を飢えさせるトリプシン処理から。 </li><li>一次および二次抗体を混ぜる。ミックス（AlexaFluor 488コンジュゲート1μL二次ニワトリ抗ヤギIgG抗体と一次抗GLUT4抗体の5μL）井戸のx数。暗闇の中で、室温で10分間インキュベートする。 </li><li>培地で希釈してインスリンの2X作業用ストックを準備します。 </li><li>細胞が付着したかしていないかどうかを確認します。 </li><li>穏やかに細胞を含むウェルにインスリンのご2X作業用ストックの6μL抗体ミックスと0.5 mLを加える。培地のいずれかの乱流を避けてください。暗所で30分間インキュベート（37℃、5％CO 2）、。 </li><li>細胞を振盪せずに、各ウェルにPBS + 1％PFAの0.5 mLを加えることによって細胞を固定する。暗闇の中で、室温で20分間インキュベートする。 </li><li>あなたの細胞がウェルに付着している場合は、軽く細胞リフターでそれらを持ち上げます。あなたのフローサイトメーターに適合して細胞をペレット化する遠心チューブに細胞を（1 mLの合計）に転送します。 </li><li> 0.4 mLのPBS + 1％PFAで1 mLのPBSに懸濁するで二回ペレットを洗浄します。この時点で、細胞はホイルでラップすることができますし、冷蔵庫に保存されている（4-8 ° C）または即時のデータ取得のためのフローサイトメーターに撮影。 </li></ol> 2。データ収集<ol><li>側方散乱光（SSC）に対する前方散乱光（FCS）パネルで、生細胞の周り広範なゲートを設定します。おFL1パラメータを設定するには、負のチューブを（アイソタイプコントロールとインスリンや細胞で処理していない細胞&quot;ステンドグラス&quot;）を使用してください。あなたのピークは、パネルで完全に下側のカットではないことを確認してください。 </li><li>チューブごとに少なくとも10,000個の細胞からデータを取得する。 </li></ol> 3。データ解析<ol><li> SSC VS FSCパネル（図1A）で生細胞の周りにゲートを描きます。 AlexaFluor 488蛍光強度に対する生細胞ゲート内の細胞（図1B）の数のヒストグラムをプロット。あなたは（図1C）の違いを視覚化する別のヒストグラムをオーバーレイが定量のために、各細胞集団の平均値と中央値蛍光強度を判断し、比較のためにこれらの番号を使用することができます。 </li><li>として図の1Cと1Dに示すようにインスリンの非存在下で得られた値にデータを正規化する。 </li></ol> 4。代表的な結果健常者（無インスリン抵抗性）から分離された筋芽細胞で、インスリンは細胞質から形質膜（図1、左）へのGLUT4の用量依存性の転座を誘発する。対照的に、インスリンは、インスリン抵抗性（図1、右）の患者から分離された筋芽細胞の形質膜へのGLUT4トランスロケーションを誘導しない。 <img src="/files/ftp_upload/2429/2429fig1.jpg" alt="図1" /> 図1。染色実験の例。の筋芽細胞がインスリン抵抗性のないドナー（左）からとインスリン抵抗性（右）患者から分離された。生細胞の周りのゲートと、SSC（側方散乱光）に対するFCS（前方散乱）プロット。B、セルはゲート、GLUT4の外部エピトープに対する抗体で染色し、そして非刺激左（赤）またはインスリン1nMの（青）、10nMの（オレンジ）、または100 nm（緑）で刺激した。図をわかりやすくするために、我々は唯一、C。インスリン抵抗性を持つ細胞に100 nMのインスリンなしとでデータを表示Bに示すようにヒストグラムの蛍光強度（MFI）の平均（無インシュリン）非刺激のまま細胞のMFIに標準化した。 D、Bに示すように、ヒストグラムから正規化されたMFIのデータのプロット

<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>

利害の衝突は宣言されません。

我々は、フローサイトメトリーにより細胞質から細胞の形質膜へのGLUT4のトランスロケーションを定量化するための迅速な技術を実証している。 GLUT4は、一時的に細胞の形質膜に発現され、エンドサイトーシスされる。結合した抗体は、さらにエンドサイトーシスの間に、GLUT4に接続されたままにするので、蛍光シグナルはインスリンの存在下でインキュベーションの選択した期間中に形質膜に露出されてGLUT4の総量を与えます。 この手法は、目的のタンパク質の外エピトープに向け良好な抗体を提供する、細胞内コンパートメントから形質膜への他の蛋白質の転座を勉強にも適用することができる利用可能です。例えば、同じようなアッセイは、現在一般的に4,5それらの形質膜にCD107aの転座を測定することにより、細胞傷害性Tリンパ球およびナチュラルキラーリンパ球の脱顆粒の割合を評価するために使用されます。細胞のアポトーシスやネクローシスの間に原形質膜の内側から外側へのホスファチジルセリンの転座はまた、蛍光標識アネキシンV 6を使用して、フローサイトメトリーにより検出することができます。 アッセイの迅速性に加え、フローサイトメトリーは、混合細胞集団における形質膜へのタンパク質転移の研究をできるという利点を提示。確かに、一つはマルチレーザーフローサイトメーター7のデータ取得、続いて細胞のサブセットマーカーのために、目的のタンパク質の染色を組み合わせることができます。 プロトコルのパラグラフ1.2で与えられた抗体の量は、出発点であり、私たちは、あなたがあなたの最大の信号を与える最小の抗体濃度を決定する目的の細胞を使用して抗体を滴定することをお勧めします。我々がここで使用している主要な抗ヒトGLUT4抗体が試験管当たり1〜10μL（チューブあたりすなわち0.2〜2μgの抗体）、これ以上の100万人以上のセルを含む各チューブになるための良い出発範囲。他の一次抗体の場合は、供給者の指示を参照してください。 細胞の自家蛍光は、ドナーからアクセプターへと実験間に変化するとして、データの正規化が必要である。

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<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

グルコースはその血中濃度の一定の規制を必要とする、身体のエネルギーの主な情報源です。膵臓からのインスリン放出は、インスリン感受性組織、特に脳、骨格筋、および脂肪細胞によるグルコース取り込みを誘導する。 2型糖尿病および/または肥満に苦しむ患者はしばしばインスリン抵抗性を開発し、そのグルコースの恒常性を制御することはできません。インスリン抵抗性のメカニズムに新しい洞察は、2型糖尿病の新しい治療戦略を提供することがあります。 グルコーストランスポーターのGLUTファミリーは、本体1全体の異なる組織に分散し13人で構成されています。グルコーストランスポータータイプ4（GLUT4）はそのような骨格筋などのインスリン感受性組織による仲介グルコースの取り込みを主要なトランスポーターです。その受容体にインスリンが結合すると、GLUT4のグルコースの取り込みを誘導、細胞質から細胞膜へ転位する含有小胞。減少GLUT4転座は、2型糖尿病2,3のインスリン抵抗性の原因の一つです。 細胞質から形質膜へのGLUT4のトランスロケーションは、蛍光標識GLUT4 -特異的抗体を用いて、免疫細胞化学により可視化することができます。 ここで、我々は、フローサイトメトリーを用いて、選択した期間中に細胞の形質膜へのGLUT4転座の合計額を定量化する手法を説明します。このプロトコルは急速である（4時間未満、インスリンとのインキュベーションを含む）と一回の実験で、わずか3000のような細胞または条件ごとに、多くの100万などの細胞の分析を行うことができます。それはそれにバインドするとすぐそれが細胞膜へトランスロケーションした後に細胞外培地にさらされるとして、そのトランスポーターの外部エピトープに向けられた抗GLUT4抗体に依存しています。

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

このプロトコルは、フローサイトメトリーにより細胞質から細胞の形質膜へのGLUT4のトランスロケーションを定量化するために迅速なテクニックを説明します。

フローサイトメトリーによる細胞膜へのGLUT4転の定量的測定

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

Research

JoVE Journal

Biology

26.3K ビュー.  Baylor College of Medicine. このプロトコルは、フローサイトメトリーにより細胞質から細胞の形質膜へのGLUT4のトランスロケーションを定量化するために迅速なテクニックを説明します。

ビデオ: 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.

リバーシブルビオチン化によって原形質膜タンパク質エンドサイトーシス率を測定

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

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：フローサイトメトリーにより検出免疫沈降

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.

マウス減数分裂細胞のフローサイトメトリーの精製

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.

複数のターゲットトレース（MTT）による原形質膜中の分子拡散のマッピング

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.

細胞トラッキング染料を使用して細胞分裂監視のための最適化された染色と増殖モデリング方法

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.

血漿レチノール結合タンパク質の膜受容体によってレチノール交通のリアルタイム解析

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.

単離されたミトコンドリアのフローサイトメトリーによる外膜タンパク質の免疫検出

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.

孤立した塊茎プロトプ ラストのフローサイトメトリーによる表現を測定

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.

蛍光レポーターの pH 依存性を用いたフローサイトメトリー流 mt 桂馬によって Mitophagy の高感度測定

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.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

フローサイトメトリーによる細胞性粘菌キイロタマホコリカビマクロピノサイトーシスの高スループット測定

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...