Diese Arbeit wurde von den Eagles Innovation Award (Fraternal Order of Eagles) und von der Mayo Foundation unterstützt.

Hochempfindliche Detektion und quantitative Analyse von nativem Protein-Protein-Interaktionen und Multiproteinkomplexen mittels Durchflusszytometrie: ** Dieses Video-Protokoll basiert auf einem zugehörigen Publikation 1. Adam G. Schrum, Diana Gil, Elaine P. Dopfer, David L. Wiest, Laurence A. Turka, Wolfgang WA Schamel, Ed Palmer. Science STKE 2007 (389): pl2, 5. Juni 2007, [DOI: 10.1126/stke.3892007pl2]. <a href="http://stke.sciencemag.org/cgi/content/abstract/2007/389/pl2">Bitte klicken Sie hier, um diese Publikation zu sehen</a> . Vor Beginn der Vorbereitung der folgenden wässrigen Stammlösungen: <table><tbody><tr><td> MES Coupling Buffer: </td><td> Lagerung bei 25 ° C </td></tr><tr><td> MES, pH 6,0 </td><td> 50 mM </td></tr><tr><td> EDTA </td><td> 1 mM </td></tr><tr><td> Abschrecken / Blocking / Storage (QBS) Buffer: </td><td> Lagerung bei 4 ° C </td></tr><tr><td> BSA </td><td> 1% </td></tr><tr><td> Natriumazid </td><td> 0,02% </td></tr><tr><td> PBS, pH 7,4 </td><td> 1x </td></tr><tr><td> FCM-Puffer: </td><td> Lagerung bei 4 ° C </td></tr><tr><td> Tris, pH 7,4 </td><td> 50 mM </td></tr><tr><td> NaCl </td><td> 100 mM </td></tr><tr><td> Natriumazid </td><td> 0,02% </td></tr><tr><td> FBS </td><td> 5% </td></tr><tr><td> PBS, pH 7,4 </td><td> 1x </td></tr></tbody></table> Bereiten Sie unmittelbar vor dem Gebrauch: <table><tbody><tr><td> EDAC-MES: </td><td> 50 mg / mL EDAC Pulver in MES Kupplungspuffer </td></tr><tr><td> Digitonin-Lösung (2% w / v): </td><td> Lösen Sie Digitonin Pulver in dH 2 O durch Erhitzen auf 95 ° C für 5min anschließendes Abkühlen auf Eis </td></tr><tr><td> Lysis Buffer: </td><td></td></tr><tr><td> Tris, pH 7,4 </td><td> 50 mM </td></tr><tr><td> NaCl </td><td> 150 mm </td></tr><tr><td> Digitonin-Lösung </td><td> 1% </td></tr><tr><td> Protease-Inhibitoren </td><td> 1x </td></tr><tr><td> Keep on ice </td><td></td></tr></tbody></table> 1. Kopplung von mAb an Beads <ol><li> Bestimmen Sie die Konzentration der Perlen aus den gekauften Aktien durch Verdünnung in PBS und das Zählen mit einem Hämacytometer. Beginnen Sie mit einer 1:10000 Verdünnung von Perlen zum Zählen. </li><li> Pipette 18 x 10 6 Perlen in einer 1,5-ml-Reaktionsgefäß. </li><li> Waschen Sie die Kugeln von 2 bis 3 mal in 0,5 bis 1,0 mL MES-Kupplung Puffer, Zentrifugieren bei 20.000 g für 3 Minuten bei 25 ° C nach jeder Wäsche. </li><li> Resuspendieren der Beads in 50 ul MES Coupling Buffer. </li><li> Aktivieren Sie die Carboxylgruppen auf die Perlen durch Zugabe von 20 ul der frisch zubereiteten EDAC-MES. </li><li> Vorsichtig mischen und 15 min bei 25 ° C von Hand-und Abpipettieren. </li><li> Waschen Sie den aktivierten Beads 2 bis 3 mal in 0,5 bis 1,0 ml PBS, Zentrifugation bei 20.000 g für 3 Minuten bei 25 ° C nach jeder Wäsche. </li><li> Resuspendieren aktivierten Beads in 50 ul PBS. </li><li> Dann werden 50 ul der IP mAb (at 0,2-1,0 mg / mL Lager-Konzentration) an die aktivierten Bead-Lösung. </li><li> Mix für 3 bis 4 Stunden bei 25 ° C, indem man das Rohr auf einer vibrierenden Schüttler. Schütteln Sie genug, um zu verhindern Absetzen der Perlen auf dem Boden des Röhrchens. </li><li> Wash mAb-gekoppelten Kügelchen von 2 bis 3 mal in 0,5 bis 1,0 ml PBS, Zentrifugation bei 20.000 g für 3 Minuten bei 25 ° C nach jeder Wäsche. </li><li> Resuspendieren der Beads in 100 ul QBS Buffer. Diese können bei 4 ° C gelagert werden </li><li> Aussetzung der Kügelchen gut, verdünnte 1:200-1:10,000 in PBS und zählen mit einem Hämacytometer. Die Konzentration muss für jeden präparativen Batch gemessen werden, so dass die Anzahl der Kugeln in jede IP-oder Pull-Down-Experiment verwendet genau gesteuert werden kann. </li></ol> 2. Post-nukleare Lysate Vorbereitung und Ip Die Lyse-Verfahren und optimale Lysebedingungen wird nach Zelltyp und Protein-Protein-Interaktionen untersucht abhängen. Eine Reihe von Protokollen vorhanden und können für Ihre Anwendung geändert werden. <ol><li> Lyse 20 x 10 6 &quot;kleine&quot; Zellen, wie Lymphozyten oder 5 x 10 6 &quot;große&quot; Zellen, wie Makrophagen oder Tumorzellen, in 100 ul frisch Lysepuffer in einem 1,5-ml Mikrozentrifugenröhrchen für 20min auf Eis. Skalieren Sie die Lyse Volumen je nach Bedarf. </li><li> Zum Entfernen Kerne und unlöslichen Zelltrümmer, Zentrifuge das Lysat bei 20.000 g für 10 min bei 4 ° C. Halten Sie den Überstand und entsorgen Sie die Pellets. </li><li> Add 0,5 x 10 5 bis 2,5 x 10 5 mAb-gekoppelten Kügelchen, um das geklärte Lysat, Pipettieren vorsichtig mischen. Das Mindestvolumen wir nutzen, umSie eine einzelne IP ist 5 ul. </li><li> Legen Sie auf einer vertikalen rotierenden Rad 4 Stunden bis über Nacht in einem kalten Raum. Set, um eine ausreichende Geschwindigkeit, um die Perlen Absetzen zu verhindern. Es ist akzeptabel für die Flüssigkeit der Low-Volume-IPs in den Boden des Röhrchens während der Rotation bleiben. </li></ol> 3. Probing der Bead-Capture-Protein mit Fluorochrom-konjugierten Antikörpern <ol><li> Waschen Sie die IP-Perlen zweimal in 0,2 bis 1,0 ml eiskaltem FCM-Puffer, Zentrifugieren bei 20.000 g bei 4 ° C für 3 min nach jeder Wäsche. </li><li> Resuspendieren der Beads in 100 ul FCM-Puffer und 20 ul Aliquot in jedem der fünf oder mehr 1,5-ml-Reaktionsgefäße oder Vertiefungen einer Kamin-Bodenplatte. </li><li> Add Fluorochrom-konjugierten mAbs zu den Proben. Führen Sie die FCM nach der Anweisungen des Herstellers oder nach empirisch ermittelten Konzentrationen Fleck. Als Ausgangspunkt, fügen Sie 0,2 bis 1 ul der Lager-Antikörper-Lösung (bei 0,2-1,0 mg / ml) pro Röhrchen oder gut, und Inkubation für 40 min auf Eis. </li><li> Wash sondiert Perlen in 1,5-ml-Röhrchen zweimal in 1,0 ml eiskaltem FCM-Puffer, Zentrifugieren bei 20.000 g bei 4 ° C für 3 min nach jeder Wäsche. Entfernen Sie vorsichtig den Überstand nach jeder Wäsche, kümmert sich nicht um die Perlen zu stören. Für einen Kamin-Bodenplatte, waschen zweimal mit 0,2 ml eiskaltem FCM-Puffer, Zentrifugation bei 1000g für 5 min bei 4 ° C nach jeder Wäsche. Ein Mehrkanalpipette ist nützlich für diesen Schritt. Zum Entfernen von Perlen in einem Kamin-Bodenplatte Überstand, &quot;Flick&quot; die Platte einmal, dann, während noch upside-down, blot keine tropft von den Rändern der Vertiefungen. Das Restvolumen in jede Vertiefung, die die Perlen, etwa 20 ul werden. </li><li> Resuspendieren der Beads in 200 ul FCM-Puffer pro Probe. Transfer zum gekennzeichnet FACS-Röhrchen. Die Proben sind nun bereit für FCM. </li></ol> 4. FCM Acquisition Die CML Perlen in diesem Protokoll beschrieben werden 3 bis 5 um im Durchmesser, etwa die Hälfte des Durchmessers eines ruhenden Maus-Lymphozyten. Daher kann es notwendig sein, manuell erhöhen die Forward Scatter (FSC) amp zu gewinnen und den Side Scatter (SSC) Spannung, um für die Bevölkerung der Raupe Ereignisse auf der Skala zu registrieren. Einzelne IP-Perlen sollten eine einzige dicht gruppierten Bevölkerung. Die Einstellungen und Tor sollte so eingestellt werden bead Dubletten und Schmutz auszuschließen. <ol><li> Vor dem Ausführen von Perlen oder Kügelchen Standards, entfernen Sie die Tropfen Containment-Hülle, die den Probeneingang umgibt auf einigen Zytometern. Lassen Sie den Mantel Flüssigkeit zwischen den Proben tropfen, da dies den Einlass klar werden und verhindern, dass Perlen aus wurden auf das von einer Probe zur anderen. </li><li> Verwenden Sie unmarkiertem Perlen, eine negative Kontrolle der Fluoreszenz, und Rainbow Calibration Partikel (RCPs), eine positive Kontrolle der Fluoreszenz, um die Einstellungen so anpassen, dass sowohl die negativen und positiven Fluoreszenz Extremen gleichzeitig auf dem log-Skala x-Achse. Dies gewährleistet, dass die Fluoreszenz der experimentellen Proben werden auch auf-Skala. Beachten Sie, dass RCPs kleiner als die CML Perlen sind, so FSC-und SSC-Parameter sowie Schieberstellung, müssen eventuell vorübergehend für diese Probe verändert werden. </li><li> Zurück zu unmarkiertem-Bead-Einstellungen einmal Kalibrierung mit RCPs abgeschlossen ist. Wenn nur eine fluoreszierende Sonde pro Probe verwendet wird, ist keine Fluoreszenz Kompensation notwendig. Im Allgemeinen Sonde wir Multiproteinkomplexen mit einem Fluorochrom-konjugierten mAb pro FCM Probe, und wir untersuchen vielfachen Wechselwirkungen Partner durch Färbung parallel Perle Proben mit mAk spezifisch für die verschiedenen Untereinheiten. </li><li> Erfassen und Speichern des Fluoreszenz-Dateien. Die Analyse kann dann unter Verwendung der Durchflusszytometrie Software wie CellQuest, FlowJo oder CFlow werden. </li></ol> 5. Repräsentative Ergebnisse <img src="/files/ftp_upload/2066/2066fig1.jpg" alt="Abbildung 1" /> Abbildung 1. IP-FCM für TCR/CD3 Multiproteinkomplex. T-Zellen aus der Maus-Stamm BALB / c wurden in 1% Digitonin lysiert und das Lysat wurde auf IP-FCM unterzogen Verwendung von Anti-CD3ε Perlen. Die erfassten Komplexe enthalten erhebliche Mengen an TCR-β (lila-Region) und Co-assoziierte CD3-ε (grün), aber nahe Hintergrundbelastung (definiert durch die irrelevant Immunglobulin-Sonde, pink trace) von anderen Proteinen, wie Thy1.2, CD45 oder H-2K (d) (braun, orange und blau Spuren bezeichnet). Siehe ähnliche bisher veröffentlichten Ergebnisse 1-6.

<ol>
	<li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+detection+and+quantitative+analysis+of+native+protein-protein+interactions+and+multiprotein+complexes+by+flow+cytometry.">High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.</a> Sci STKE. , pl2-pl2 (2007).</li><li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+multiprotein+complexes+by+flow+cytometry.">Visualization of multiprotein complexes by flow cytometry.</a> Curr Protoc Immunol. Chapter 5, 9-9 (2009).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+division+and+death+are+segregated+by+mutation+of+TCRbeta+chain+constant+domains.">T cell division and death are segregated by mutation of TCRbeta chain constant domains.</a> Immunity. 21, 515-526 (2004).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+T+Cell+receptor+signals+determine+CD8++memory+versus+effector+development.">Different T Cell receptor signals determine CD8+ memory versus effector development.</a> Science. 323, 502-505 (2009).</li><li>Treves, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctate+is+a+key+element+in+calcium+entry+induced+by+activation+of+InsP3+receptors+and/or+calcium+store+depletion.">Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion.</a> J Cell Biol. 166, 537-548 (2004).</li><li>Gil, D., Schrum, A. G., Alarcon, B., Palmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+engagement+by+peptide-MHC+ligands+induces+a+conformational+change+in+the+CD3+complex+of+thymocytes.">T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 complex of thymocytes.</a> J Exp Med. 201, 517-522 (2005).</li></ol>

Informationen über Protein-Protein-Wechselwirkungen ist äußerst relevant für die Analyse von vielen zellulären Prozessen wie Signaltransduktion, Abstammung Reifung, Zellzyklus-Progression und Apoptose-Kaskaden. IP-FCM bietet eine schnelle, quantitative und sensible Art der Wechselwirkung von Proteinen zu untersuchen und zu definieren Mitglieder Multiproteinkomplexen in nativer Konformation. Beads gekoppelt und inkubiert mit Zelllysaten in einem Tag und überprüft werden kann und analysiert den nächsten Tag. Ein 96-Well-Platte Format ermöglicht einer großen Anzahl von Proben, die gleichzeitig eine effiziente Datenerfassung für statistische oder Screening-Zwecke bieten analysiert werden. Unter Verwendung von fluoreszierenden Kügelchen Standards, kann die Anzahl der Proteine, die durch jede Perle eingefangen geschätzt werden. Sehr wenig Ausgangsmaterial für die Erfassung und Erkennung benötigt wird, so beschränkt Proben und knappen Analyten kann noch für mehrere Interaktionen analysiert werden. Obwohl IP-FCM nicht erforderlich ist Gentechnik, Epitop-Tagging, Denaturierung oder in-vitro Mischen von Proteinen in einer nicht-physiologischen Umgebung, kann es mit diesen und anderen Techniken gekoppelt werden, wodurch es zu einem wertvollen und zugänglichen Tool mit Anwendbarkeit auf vielen biologischen Systemen. Fehlerbehebung: Viele IP-FCM Experimente erzeugen nützliche Protein-Interaktion Daten sie das erste Mal versucht werden. Allerdings kann die Optimierung von IP-FCM Antikörperkonjugation zu Perlen, Protein-Komplex zu erfassen, und fluoreszierende Sonde verbindlich zu verbessern. Die Effizienz von IP Antikörperkonjugation kann durch Sondieren gekoppelt Perlen direkt mit einem anti-Immunglobulin-Antikörper bestimmt werden. Wenn dies die Effizienz gering ist, kann die Erhöhung der Konzentration von Antikörpern während der Kupplung ermöglicht mehrere IP-Antikörper an jeder Perle befestigen. Dies kann die Bindungskapazität der IP bead Charge, was zu einer erhöhten Aufnahme und Detektion von Analyten. Andere primäre-Amin-haltigen Molekülen (z. B. Tris, Rinderserumalbumin) sollte nicht während der Kupplung vorhanden ist, da diese mit dem mAb für bead Befestigung und führen zu niedrigen mAb Kopplung konkurrieren. Wenn die Konjugation von mAb an Perlen als problematisch erweist aus anderen Gründen, können Perlen statt an Avidin / Streptavidin gekoppelt werden, und biotinylierte Antikörper kann anschließend nicht-kovalent gebunden und für die Immunpräzipitation. Trotz guter Antikörperkonjugation kann anfänglichen Erkennung von bead-assoziierten Fluoreszenz gering sein. Zunächst komplexen Erfassung selbst kann niedrig sein. Da IP-FCM hängt von der Konzentration von Analyten, die Erhöhung der Anzahl der Zellen pro Lyse Volumen lysiert erhöhen Analyt-Abscheidung und-Erkennung. Darüber hinaus kann Gefangennahme durch die Verringerung der Anzahl der IP-Kügelchen, die mit dem Lysat, die gefangen genommen Komplexe in weniger Perlen und führt zu einer erhöhten durchschnittlichen Fluoreszenz pro Bead wenn sondiert vertreibt inkubiert verbessert werden. Zweitens ist es möglich, dass der Zugang der Sonde Antikörper, um das aufgenommene Komplexe sterisch durch die immunoprecipitating Antikörper behindert. In diesem Fall kann das Problem durch den Einsatz verschiedener Antikörper und / oder die Sonde erfassen angesprochen werden.

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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>carboxylate-modified polystyrene latex (CML) beads (5mm surfactant-free)</td>
<td>Reagent</td>
<td>Interfacial Dynamics Corporation/Molecular Probes</td>
<td>2-5000</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>Rainbow Calibration Particles</td>
<td>Reagent</td>
<td>Spherotech, Inc.</td>
<td>RCP-30-5A</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>2-[N-Morpholino] ethanesulfonic acid (MES)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M-5287</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3-(3-dimethylaminopropl) carbodiimide HCl (EDAC)</td>
<td>Reagent</td>
<td>Pierce Sigma</td>
<td>22980 E-6383</td>
<td>Store at -20&deg;C under desiccating conditions.</td>
</tr>
<tr>
<td>Protease Inhibitor General Use Cocktail, containing AEBSF, Bestatin, Aprotinin, EDTA, E-64, and Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-2714</td>
<td> Prepare 100x stock solution in H2O. Aliquot and store at -20&deg;C. Handle with gloves.</td>
</tr>
<tr>
<td>PCR plates, 96-well 0.2mL, natural, tall chimney</td>
<td>Reagent</td>
<td>Fisher</td>
<td>08-408-230</td>
<td>For use in place of microcentrifuge tubes during FCM staining</td>
</tr>		</tbody>
		</table>
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ip-fcm:  immunoprecipitation detected by flow cytometry

Immunpräzipitation mittels Durchflusszytometrie (IP-FCM) erkannt ist eine effiziente Methode zur Detektion und Quantifizierung von Protein-Protein-Interaktionen. Das Grundprinzip erstreckt, dass der Sandwich-ELISA, wobei die erfassten primären Analyten zusammen mit anderen Molekülen physisch innerhalb Multiproteinkomplexen verbunden erkannt werden. Das Verfahren beinhaltet die kovalente Kopplung von Polystyrol-Latex-Mikroperlen mit immunoprecipitating monoklonale Antikörper (mAb) spezifisch für ein Protein von Interesse, Inkubation diese Perlen mit Zelllysaten, Sondieren erfasst Protein-Komplexen mit Fluorochrom-konjugierten Sonden und Analyse bead-assoziierten Fluoreszenz mittels Durchflusszytometrie. IP-FCM ist extrem empfindlich, ermöglicht die Analyse der Proteine ​​in ihrer nativen (nicht-denaturierten) Zustand, und ist zugänglich entweder semi-quantitative oder quantitative Analyse. Als weitere Vorteile, erfordert IP-FCM keine Gentechnik oder spezielle Ausrüstung, außer einem Durchflusszytometer, und es kann ohne weiteres für High-Throughput-Anwendungen angepasst werden.

Watch this Scientific Journal Video about IP-FCM:  Immunoprecipitation Detected by Flow Cytometry at JoVE.com

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Die IP-FCM-Methode vorgestellt, die eine sensible, robust, biochemische Beurteilung der nativen Protein-Protein-Interaktionen ermöglicht, ohne dass Gentechnik oder große Stichproben.

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 ...

ip-fcm-immunoprecipitation-detected-by-flow-cytometry

Research

JoVE Journal

Biology

17.4K Aufrufe.  Mayo Clinic. Die IP-FCM-Methode vorgestellt, die eine sensible, robust, biochemische Beurteilung der nativen Protein-Protein-Interaktionen ermöglicht, ohne dass Gentechnik oder große Stichproben.

Serie: IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

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

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

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 ...

Forschung

Biologie

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 ...

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 ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Cytosolischen Calcium Messungen in Nierenepithelzellen mittels Durchflusszytometrie

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

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 ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

High Efficiency Differenzierung von humanen pluripotenten Stammzellen zu Kardiomyozyten und Charakterisierung mittels Durchflusszytometrie

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED Thermo Flow - Die Kombination mit Optogenetik Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analyse von Zellsuspensionen isoliert von festen Geweben von Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

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.

Messung der Endoreduplikation durch Durchflusszytometrie von isolierten Knolle Protoplasten

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

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 ...