Diese Arbeit wurde durch Zuschüsse der Mrs. Clifford Elder Weiß Graham unterstützte Stiftungsprofessur Research Fund und die NIH (AR059838) zu CB, von der NIH (AR052791, AR044387-ARRA, &amp; NS063298) zu LTT, und von der Baylor College of Medicine Cytometry und Cell Sorting-Core mit Mitteln aus dem NIH (NCRR S10RR024574, NIAID AI036211, und NCI P30CA125123). Der Inhalt ist ausschließlich in der Verantwortung der Autoren und stellen nicht notwendigerweise die offizielle Meinung des NIH.

1. Zellfärbung <ol><li> Bereiten Sie die Zellen. Die Zellen sollten verwendet werden, während immer noch aktiv wachsende (60 bis 80% Konfluenz) werden. Serum hungern die Zellen über Nacht, Teller ihnen Zellen bei 0,1 Millionen pro Well in 0,5 ml serum-freiem Medium in einer 24-Well-Platte und in den Inkubator (37 ° C, 5% CO 2) für 30 min - 2 Stunden zu erholen von Trypsinierung. </li><li> Mischen Sie die primären und sekundären Antikörper. Mix (5 ul des primären anti-GLUT4 Antikörper mit 1 ul sekundären Huhn anti-Ziege IgG-Antikörper, konjugiert mit AlexaFluor 488) x Anzahl der Brunnen. Inkubieren für 10 Minuten bei Raumtemperatur im Dunkeln. </li><li> Bereiten Sie eine 2X arbeiten Lager von Insulin durch Verdünnen in Medium. </li><li> Prüfen Sie, ob die Zellen eingehalten haben oder nicht. </li><li> Gently add 6 ul Antikörper-Mix und 0,5 mL von 2X arbeiten Bestand an Insulin, um den Brunnen mit den Zellen. Vermeiden Sie jegliche Turbulenzen des Mediums. Inkubieren (37 ° C, 5% CO 2) für 30 Minuten im Dunkeln. </li><li> Fix die Zellen durch Zugabe von 0,5 ml PBS + 1% PFA in jede Vertiefung, ohne Schütteln der Zellen. Inkubieren für 20 Minuten bei Raumtemperatur im Dunkeln. </li><li> Wenn Ihre Zellen in die Vertiefungen haften geblieben sind, heben sie mit einer Zelle Heber. Übertragen Sie die Zellen (1 ml gesamt) in Röhrchen, die in Ihrem Durchflusszytometer fit und Zentrifuge die Zellen zu pelletieren. </li><li> Waschen Sie die Pellet zweimal mit 1 mL PBS resuspendieren und in 0,4 ml PBS + 1% PFA. An dieser Stelle Zellen können in Folie gewickelt und im Kühlschrank aufbewahrt (4-8 ° C) oder getroffen werden, um das Durchflusszytometer für die sofortige Datenerfassung. </li></ol> 2. Data Acquisition <ol><li> Legen Sie ein breites Tor in die lebenden Zellen in der side scatter (SSC) versus forward scatter (FCS) Panel. Verwenden Sie einen negativen Rohr (Zellen, die nicht mit Insulin oder Zellen &quot;befleckt&quot; mit einem Isotyp-Kontrolle) behandelt, um Ihre FL1-Parameter. Vergewissern Sie sich, Gipfel ist vollständig in das Panel, nicht auf der unteren Seite abgeschnitten. </li><li> Erwerben Sie Daten von mindestens 10.000 Zellen pro Röhrchen. </li></ol> 3. Data Analysis <ol><li> Zeichnen Sie einen Schutzzaun um die lebenden Zellen in der SSC vs FSC-Panel (Abbildung 1A). Zeichnen Sie die Histogramme der AlexaFluor 488 Fluoreszenzintensität im Vergleich zur Anzahl von Zellen innerhalb des Live-cell-Tor (Abbildung 1B). Sie können Overlay verschiedenen Histogramme, um Unterschiede zu visualisieren, sondern für die Quantifizierung, die mittlere und die mediane Fluoreszenzintensität für jede Zelle Bevölkerung und der Nutzung dieser Nummern für Ihre Vergleiche (Abbildung 1C). </li><li> Normalisieren der Daten, um den Wert in Abwesenheit von Insulin erhalten, wie in den Abbildungen 1C und 1D gezeigt. </li></ol> 4. Repräsentative Ergebnisse In Myoblasten aus einem gesunden Menschen (keine Insulinresistenz) isoliert, induziert Insulin eine Dosis-abhängige Translokation von GLUT4 aus dem Zytoplasma an die Plasmamembran (Abb. 1, links). Im Gegensatz dazu wird Insulin nicht induzieren GLUT4 Translokation an die Plasmamembran von Myoblasten von einem Patienten mit Insulinresistenz (Abbildung 1, rechts) isoliert. <img src="/files/ftp_upload/2429/2429fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Beispiel für eine Färbung zu experimentieren. Myoblasten wurden von einem Spender, ohne Insulin-Resistenz (links) und von einem Patienten mit Insulinresistenz (rechts) isoliert. A, SSC (side scatter) versus FCS (forward scatter) Grundstück mit einem Tor in die lebenden Zellen. B, gated Cells in A, mit dem Antikörper gegen ein externes Epitop des GLUT4 gefärbt, und links unstimulierten (rot) oder mit Insulin stimuliert 1 nM (blau), 10 nM (orange) oder 100 nm (grün). Für Figur Klarheit, zeigen wir nur Daten ohne und mit 100 nM Insulin für die Zellen mit einer Insulinresistenz. C, mittleren Fluoreszenzintensitäten (MFI) der Histogramme in B gezeigt wurden, die MFI der Zellen normalisiert links unstimulierten (kein Insulin). D, Plots der normalisierten MFI Daten aus der Histogramme in B gezeigt

<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>

Wir haben ein schnelles Verfahren zur Quantifizierung der Translokation von GLUT4 aus dem Zytoplasma an die Plasmamembran von Zellen mittels Durchflusszytometrie nachgewiesen. GLUT4 ist nur vorübergehend an der Plasmamembran von Zellen exprimiert wird und durch Endozytose. Da die gebundenen Antikörper an der GLUT4, auch während der Endozytose bleiben, gibt das Fluoreszenzsignal der Gesamtbetrag der GLUT4, die an der Plasmamembran während der gewählten Dauer der Inkubation in Gegenwart von Insulin ausgesetzt war. Diese Technik kann auch zur Untersuchung der Translokation von anderen Proteinen aus einem intrazellulären Kompartiment zur Plasmamembran angewandt werden, sofern eine gute Antikörper gerichtet an einen externen Epitop des Proteins von Interesse ist vorhanden. Zum Beispiel, ist ein ähnlicher Test jetzt häufig verwendet, um die Rate der Degranulation von zytotoxischen T-Lymphozyten und natürliche Killer-Lymphozyten durch die Messung der Translokation von CD107a ihrer Plasmamembran 4,5 zu bewerten. Die Translokation von Phosphatidylserin von der Innenseite der Plasmamembran auf die Außenseite während Apoptose oder Nekrose kann auch mittels Durchflusszytometrie detektiert werden, mit Fluorophor-markierten Annexin V 6. Neben Schnelligkeit des Tests präsentiert Durchflusszytometrie den Vorteil, dass die Untersuchung von Protein-Translokation an die Plasmamembran in gemischten Zellpopulationen. Tatsächlich kann man Färbung für Zell-Untergruppe Marker und für das Protein von Interesse, durch die Datenerfassung auf einem Multi-Laser-Durchflusszytometer 7 an zu kombinieren. Die Mengen von Antikörpern in Absatz 1.2 des Protokolls gegeben sind ein Ausgangspunkt, empfehlen wir Ihnen titrieren Ihre Antikörper mit Zellen von Interesse, um die minimale Antikörperkonzentration, die Ihnen ein maximales Signal zu bestimmen. Ein guter Ausgangspunkt Bereich für die Primär-Anti-Human-Antikörper GLUT4 wir hier verwendet haben würde 1-10 ul pro Röhrchen (dh 0,2-2 ug Antikörper pro Röhrchen), wobei jedes Rohr mit nicht mehr als 1 Million Zellen. Für andere primäre Antikörper, die Anweisungen des Herstellers beziehen. Die Normalisierung der Daten ist notwendig, da die Autofluoreszenz von Zellen vom Spender zum Spender und von Experiment zu Experiment variieren.

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		<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

Glukose ist der wichtigste Energiequelle für den Körper, die eine konstante Regelung seiner Konzentration im Blut. Insulinfreisetzung der Bauchspeicheldrüse verursacht Glukoseaufnahme durch Insulin-sensitiven Geweben, vor allem das Gehirn, Skelettmuskel und Adipozyten. Patienten mit Typ-2-Diabetes und / oder Übergewicht entwickeln häufig eine Insulinresistenz und sind unfähig, ihre Glukose-Homöostase zu kontrollieren. Neue Einblicke in die Mechanismen der Insulinresistenz können neue Therapiestrategien bei Typ-2-Diabetes. Die GLUT-Familie Glucose-Transporter besteht aus dreizehn Mitgliedern auf verschiedenen Gewebe im gesamten Körper 1 verteilt. Glucose-Transporter Typ 4 (GLUT4) ist der wichtigste Transporter, vermittelt Glukoseaufnahme durch Insulin empfindlicher Gewebe wie die Skelettmuskulatur. Bei der Bindung von Insulin an seinen Rezeptor, Vesikel mit GLUT4 aus dem Zytoplasma transloziert zur Plasmamembran, induzieren Glukoseaufnahme. Reduzierte GLUT4 Translokation ist eine der Ursachen der Insulinresistenz bei Typ-2-Diabetes 2,3. Die Translokation von GLUT4 aus dem Zytoplasma an die Plasmamembran visualisiert werden können Immunzytochemie, mit Fluorophor-konjugierten GLUT4-spezifischen Antikörpern. Hier beschreiben wir eine Technik, um Gesamtmengen von GLUT4 Translokation an die Plasmamembran von Zellen in einem ausgewählten Zeitraum zu quantifizieren, mittels Durchflusszytometrie. Dieses Protokoll ist schnell (weniger als 4 Stunden, einschließlich der Inkubation mit Insulin) und ermöglicht die Analyse von so wenig wie 3.000 Zellen oder so viele wie 1 Million Zellen pro Bedingung in einem einzigen Experiment. Es stützt sich auf anti-GLUT4 Antikörper, die gegen einen externen Epitop des Transporters, die an sie bindet, sobald sie an die extrazelluläre Medium nach Translokation an die Plasmamembran ausgesetzt ist.

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

Dieses Protokoll beschreibt ein schnelles Verfahren, um die Translokation von GLUT4 aus dem Zytoplasma zu quantifizieren, um die Plasmamembran von Zellen mittels Durchflusszytometrie.

Quantitative Messung der GLUT4 Translokation an die Plasmamembran mittels Durchflusszytometrie

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 Aufrufe.  Baylor College of Medicine. Dieses Protokoll beschreibt ein schnelles Verfahren, um die Translokation von GLUT4 aus dem Zytoplasma zu quantifizieren, um die Plasmamembran von Zellen mittels Durchflusszytometrie.

Serie: 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.

Mess-Plasmamembranprotein endocytischen Preise von Reversible Biotinylierung

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

Forschung

Biologie

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: Immunpräzipitation mittels Durchflusszytometrie Detected

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.

Durchflusszytometrie Reinigung der Maus meiotischen Zellen

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.

Mapping molekulare Diffusion in der Plasmamembran durch 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 ...

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.

Optimiert Färbung und Proliferation Modeling-Methoden für die Zellteilung Überwachung mit 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 ...

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.

Real-time Analyse von Retinol Transport durch die Membran-Rezeptor von 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 ...

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.

Immundetektion von Außenmembranproteinen mittels Durchflusszytometrie von isolierten Mitochondrien

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

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.

Empfindliche Messung des Mitophagy von Flow Cytometry Verwendung der pH-abhängigen fluoreszierende Reporter Mt-Otamegane

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.

Hochdurchsatz-Messung von Dictyostelium Discoideum Macropinocytosis von Flow Cytometry

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

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.