Diese Arbeit wurde durch Fördermittel National Health and Medical Research Council of Australia (NHMRC) (526615 und 545906) und zum Teil durch die Regierung von Victoria die OIS-Programm unterstützt. DS ist ein Fellow der NHMRC.

1. Herstellung von [3 H]-Cholesterin <ol><li> In der Dunstabzug, verzichten die erforderliche Menge an [3 H] Cholesterin in ein 1,5 ml Mikrozentrifugenröhrchen (0,5 uCi (19 kBq) pro Vertiefung ist für einen typischen Test erforderlich). </li><li> Wenn [3H]-Cholesterin in Toluol suspendiert wird, trocknen Sie ihn nach unten komplett mit N2-Gas-und resuspendieren mit 100% Ethanol bis zu einer Endkonzentration von 1 uCi (37 kBq / ul. Vortex und gut mischen. </li></ol> 2. Plattieren von Zellen und Kennzeichnung zellulärem Cholesterin Dieses Protokoll wurde getestet mit folgenden Zelltypen: menschliche Monozyten 4,5, THP-1-menschlichen Monozyten-Makrophagen 6,7,8, RAW 264.7 murine Makrophagen 5,9,10,11, HeLa-Zellen 12, HUVEC (HUVEC), BHK-21 Zellen 9, Human-und Maus-Fibroblasten 13,14, HepG2 menschlichen Hepatokarzinomzellen 15 und, in modifizierter Form, von Thrombozyten &lt;sup&gt; 16. <ol><li> Die Zellen und zähle sie. Tafel Zellen in 12-Well-Platten bei der Enddichte von 0.2x10 6 Zellen pro Well in 0,9 ml Vollmedium. Die Zellen werden weiter wachsen und 0.8x10 6 Zellen pro Well zu dem Zeitpunkt der Ausströmung Experiment erreicht. Für Zellen, die sich nicht teilen, wie differenzierte THP-1 Zellen, sollte die Aussaatdichte 0.8x10 6 Zellen pro Vertiefung sein. Bei Verwendung von 6 - oder 24-Well-Platten, doppelt oder halb so viele Zellen pro Well. Die Zahl der Brunnen sollte ausreichend sein für Vierfachbestimmungen Für jeden experimentellen Zustand. </li><li> Wenn die Differenzierung von THP-1-Zellen erforderlich ist, fügen PMA (Endkonzentration 0,1 g / ml) zu den Medien für 48-72 h. </li><li> Fügen Sie eine kleine Teilmenge von Medien in das Mikrozentrifugenröhrchen mit der [3 H] Cholesterin. </li><li> Waschen Seiten der Röhre mit Medien vor dem Hinzufügen [3 H] Cholesterin in einen separaten Aliquot komplette Medien (100 ul / Well). </li> &lt;li&gt; In der Medien, die [3 H]-Cholesterin in die Vertiefungen mit Zellen (Endvolumen pro Well 1 ml). Wenn keine Behandlung der Zellen vor der Markierung erforderlich ist, können Zellen in [3 H]-Cholesterin-Medium ausplattiert werden. </li><li> Inkubieren Zellen für 48 Stunden in der Zellkultur Inkubator (37 ° C, 5% CO 2). </li></ol> 3. Äquilibrierung Incubation <ol><li> Nach 48 Stunden Inkubation, überprüfen Zellen unter dem Mikroskop. Stellen Sie sicher, sie sind gesund und sind bei etwa 80% Konfluenz. </li><li> Entfernen Sie das Medium mit [3 H] Cholesterin. Waschen Sie die Zellen vorsichtig mit PBS. Wiederholen Sie 3 mal Waschen. </li><li> Bereiten serumfreien Medien. Falls erforderlich, aktivieren Zellen durch Zugabe von LXR-Agonisten (z. B. TO-901317 bei 1-4 mmol / l) oder cAMP (0,3 mmol / l; für Zellen der Maus Ursprungs) zu serumfreien-Medien. </li><li> Fügen Sie 500 ul serumfreiem Medium in jede Vertiefung. </li><li> Inkubieren für 18 Stunden in der Zellkultur Inkubator (37 ° C, 5% CO 2). </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Cholesterin Efflux Incubation <ol><li> Nach 18 Stunden Inkubation in Serum-freiem Medium, zu überprüfen Zellen unter dem Mikroskop. Stellen Sie sicher, dass sie gesund sind und konfluent (mindestens 80% Konfluenz). </li><li> Planen Lösung von Cholesterin Efflux Akzeptoren in serumfreiem Medium. Beispiele für Akzeptoren schließen ein: Apolipoprotein AI (ApoA-I) (Endkonzentration 10 ug / ml) High Density Lipoprotein (HDL) (Endkonzentration 20 ug / ml) Cyclodextrin (Endkonzentration 200 ug / ml) Plasma (Endkonzentration 1-2%) </li><li> Waschen Sie die Zellen vorsichtig mit PBS. </li><li> Fügen Sie 250 ul serumfreiem Medium mit Akzeptoren in jede Vertiefung. </li><li> Lassen Sie eine Reihe von Brunnen, um Hintergrund-Efflux (Ausstrom zu serumfreien Medien ohne Akzeptor) bestimmen </li><li> Inkubieren Zellen für 2 Stunden in der Zellkultur Inkubator (37 ° C, 5% CO 2). Dauer der Inkubation kann Efflux von 30 min bis 8 h variieren, wenn erforderlich. </li></ol> 5. Processing SAmples <ol><li> Nach 2 Stunden Inkubation überprüfen Zellen unter dem Mikroskop. </li><li> Sammeln Sie Medien in 1,5 ml Mikrozentrifugenröhrchen. Zentrifugieren bei 14.000 rpm für 1-10 min bei Raumtemperatur, um Zelltrümmer zu entfernen. </li><li> Übertragen Sie 100 ul Medium in 7 ml Szintillations-Fläschchen. In 5 ml Insta-Gel Plus (PerkinElmer) und Vortex-Mischung. Bewahren restlichen Proben bei 4 ° C </li><li> Platzieren Sie Platten in Gefrierschrank für 30 Minuten. Fügen Sie 500 ul dH 2 O in jede Vertiefung. Prüfen Zellen unter dem Mikroskop zu gewährleisten, dass alle Zellen von der Unterseite der Vertiefungen angehoben. Wenn noch anhaftenden, entweder die Platten mit dH 2 O bei 4 ° C über Nacht oder Kratzen Brunnen. </li><li> Wenn alle Zellen aufgehoben wurden ausgeschaltet, und Abpipettieren zum Aufbrechen Zellklumpen. Übertragen Sie 100 ul in 7 ml Szintillationsgefäße. In 5 ml Insta-Gel Plus (PerkinElmer) und Vortex-Mischung. Bewahren restlichen Platten bei 4 ° C </li><li> Platzieren Sie Szintillationsgefäße auf Szintillationszähler. Konfigurieren Zähler für [3 H] zählen indpm Einheiten. </li></ol> 6. Die Analyse der Ergebnisse <ol><li> Die Rate der Cholesterin-Efflux in der Regel als ein Anteil von Cholesterin aus den Zellen verschoben auf den Akzeptor ausgedrückt. Die folgende Formel verwendet wird: </li></ol><img alt="Gleichung 1" src="/files/ftp_upload/3810/3810eq1.jpg" /><ol start="2"><li> Die spezifische Abgabe erfolgt als eine Differenz zwischen den Ausfluß in Gegenwart oder Abwesenheit des Akzeptors (leer) berechnet. </li></ol><img alt="Gleichung 2" src="/files/ftp_upload/3810/3810eq2.jpg" /> 7. Timeline <img alt="1" src="/files/ftp_upload/3810/3810timeline.jpg" /> 8. Repräsentative Ergebnisse Ein Beispiel für ein Ergebnis einer Cholesterin-Efflux Versuchs ist in gezeigt. 1. In diesem Experiment THP-1 Monozyten wurden in Makrophagen und Cholesterin Efflux auf verschiedene Akzeptoren differenziert wargetestet. Cholesterin Efflux um Medium ohne Akzeptoren (&quot;blank&quot;) betrug 0,79% und dieser Wert wurde als nicht-spezifische Efflux angesehen und von anderen Werten abgezogen. Cholesterin Efflux für die menschliche ApoA-I (Endkonzentration 30 ug / ml) war 4,75%. Ein Verweis Plasmaprobe wurde in diesem Experiment aufgenommen, um inter-experimentellen Variabilität überwachen. Eine Referenzprobe kann für die Normalisierung der Daten über eine große Anzahl von Experimenten verwendet werden, aber wir fanden es sinnvoll, den Test wiederholen, wenn die Variabilität ist hoch. Ein Patient Plasma (Endkonzentration 2%) wurde getestet, bevor und nachdem der Patient mit Medikamenten behandelt wurde. Es wurde gefolgert, dass bei diesem Patienten Medikamente hatten einen negativen Einfluss auf die Kapazität des Plasma-Cholesterin-Efflux zu unterstützen. Ein weiteres Beispiel für ein Ergebnis des Experiments ist in Efflux gezeigt. 2. In diesem Experiment wurden Makrophagen-RAW 264.7 aktiviert oder nicht aktiviert durch Inkubation über Nacht mit LXR-Agonisten TO-901.317 (Endkonzentration1 mol / L) und Cholesterin Efflux zu der gleichen Probe von menschlichem Plasma (2%) wurde getestet. Es wurde festgestellt, dass die Aktivierung der zellulären Expression von ABC-Transportern mit LXR-Agonisten die Fähigkeit von Zellen, Cholesterin zu extrazellulären Akzeptor freizugeben erhöht. <img alt="1" src="/files/ftp_upload/3810/3810fig1.jpg" /> Abbildung 1. Cholesterin Efflux aus THP-1 Zellen auf verschiedene Akzeptoren. Anteil von Cholesterin Efflux (dh der Anteil der markierten Cholesterin aus den Zellen bewegt, um die angegebene Akzeptor) nach Abzug des Rohlings Werte beschränkt. Mittelwert ± Standardabweichung von Vierfachbestimmungen, * p &lt;0,05. <img alt="2" src="/files/ftp_upload/3810/3810fig2.jpg" /> Abbildung 2. Cholesterin Ausfluß aus RAW 264.7 aktiviert oder nicht aktiviert mit LXR-Agonisten an menschlichem Plasma. Anteil von Cholesterin Efflux (dh der Anteil der markierten Cholesterin aus den Zellen bewegt, um dieangegeben Akzeptor) wird nach Abzug der Blindwerte gezeigt. Mittelwert ± SD von Vierfachbestimmungen, * p &lt;0,001.

<ol><li>Tabas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cholesterol+in+health+and+disease%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Cholesterol in health and disease.</a> J. Clin. Invest. 110, 583-590 (2002).</li>
<li>Brown, M. S., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cholesterol+feedback%3A+from+Schoenheimer%27s+bottle+to+Scap%27s+MELADL%5BTitle%5D)+AND+%22J.+Lipid+Res%22%5BJournal%5D)">Cholesterol feedback: from Schoenheimer's bottle to Scap's MELADL.</a> J. Lipid Res. 50, 15-27 (2009).</li>
<li>Tabas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Consequences+of+cellular+cholesterol+accumulation%3A+basic+concepts+and+physiological+implications%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Consequences of cellular cholesterol accumulation: basic concepts and physiological implications.</a> J. Clin. Invest. 110, 905-911 (2002).</li>
<li>Murphy, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-Density+Lipoprotein+Reduces+the+Human+Monocyte+Inflammatory+Response%5BTitle%5D)+AND+%22Arterioscler.+Thromb.+Vasc.+Biol%22%5BJournal%5D)">High-Density Lipoprotein Reduces the Human Monocyte Inflammatory Response.</a> Arterioscler. Thromb. Vasc. Biol. 28, 2071-2077 (2008).</li>
<li>Mujawar, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+Immunodeficiency+Virus+Impairs+Reverse+Cholesterol+Transport+from+Macrophages%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">Human Immunodeficiency Virus Impairs Reverse Cholesterol Transport from Macrophages.</a> PLoS Biology. 4, 365-36 (2006).</li>
<li>Patel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reconstituted+High-Density+Lipoprotein+Increases+Plasma+High-Density+Lipoprotein+Anti-Inflammatory+Properties+and+Cholesterol+Efflux+Capacity+in+Patients+With+Type+2+Diabetes%5BTitle%5D)+AND+%22J.+Am.+Coll.+Cardiol%22%5BJournal%5D)">Reconstituted High-Density Lipoprotein Increases Plasma High-Density Lipoprotein Anti-Inflammatory Properties and Cholesterol Efflux Capacity in Patients With Type 2 Diabetes.</a> J. Am. Coll. Cardiol. 53, 962-971 (2009).</li>
<li>Shaw, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infusion+of+reconstituted+high-density+lipoprotein+leads+to+acute+changes+in+human+atherosclerotic+plaque%5BTitle%5D)+AND+%22Circ.+Res%22%5BJournal%5D)">Infusion of reconstituted high-density lipoprotein leads to acute changes in human atherosclerotic plaque.</a> Circ. Res. 103, 1084-1091 (2008).</li>
<li>Kekulawala, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+freezing+on+high-density+lipoprotein+functionality%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">Impact of freezing on high-density lipoprotein functionality.</a> Anal. Biochem. 379, 75-77 (2008).</li>
<li>D'Souza, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure%2FFunction+Relationships+of+Apolipoprotein+A-I+Mimetic+Peptides%3A+Implications+for+Antiatherogenic+Activities+of+High-Density+Lipoprotein%5BTitle%5D)+AND+%22Circ.+Res%22%5BJournal%5D)">Structure/Function Relationships of Apolipoprotein A-I Mimetic Peptides: Implications for Antiatherogenic Activities of High-Density Lipoprotein.</a> Circ. Res. 107, 217-227 (2010).</li>
<li>Tchoua, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+cholesteryl+ester+transfer+protein+overexpression+and+inhibition+on+reverse+cholesterol+transport%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">The effect of cholesteryl ester transfer protein overexpression and inhibition on reverse cholesterol transport.</a> Cardiovasc Res. 77, 732-739 (2008).</li>
<li>Mukhamedova, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancing+apolipoprotein+A-I-dependent+cholesterol+efflux+elevates+cholesterol+export+from+macrophages+in+vivo%5BTitle%5D)+AND+%22J.+Lipid+Res%22%5BJournal%5D)">Enhancing apolipoprotein A-I-dependent cholesterol efflux elevates cholesterol export from macrophages in vivo.</a> J. Lipid Res. 49, 2312-2322 (2008).</li>
<li>Mukhamedova, N., Fu, Y., Bukrinsky, M., Remaley, A. T., Sviridov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Role+of+Different+Regions+of+ATP-Binding+Cassette+Transporter+A1+in+Cholesterol+Efflux%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">The Role of Different Regions of ATP-Binding Cassette Transporter A1 in Cholesterol Efflux.</a> Biochemistry. 46, 9388-9398 (2007).</li>
<li>Sviridov, D., Fidge, N., Beaumier-Gallon, G., Fielding, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apolipoprotein+A-I+stimulates+the+transport+of+intracellular+cholesterol+to+cell-surface+cholesterol-rich+domains+%28caveolae%5BTitle%5D)+AND+%22Biochem.+J%22%5BJournal%5D)">Apolipoprotein A-I stimulates the transport of intracellular cholesterol to cell-surface cholesterol-rich domains (caveolae.</a> Biochem. J. 358, 79-86 (2001).</li>
<li>Smyth, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mouse+model+of+harlequin+ichthyosis+delineates+a+key+role+for+abca12+in+lipid+homeostasis%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">A mouse model of harlequin ichthyosis delineates a key role for abca12 in lipid homeostasis.</a> PLoS Genet. 4, e1000192-e1000192 (2008).</li>
<li>Fu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+Caveolin-1+Enhances+Cholesterol+Efflux+in+Hepatic+Cells%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Expression of Caveolin-1 Enhances Cholesterol Efflux in Hepatic Cells.</a> J. Biol. Chem. 279, 14140-14146 (2004).</li>
<li>Calkin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reconstituted+High-Density+Lipoprotein+Attenuates+Platelet+Function+in+Individuals+With+Type+2+Diabetes+Mellitus+by+Promoting+Cholesterol+Efflux%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Reconstituted High-Density Lipoprotein Attenuates Platelet Function in Individuals With Type 2 Diabetes Mellitus by Promoting Cholesterol Efflux.</a> Circulation. 120, 2095-2104 (2009).</li>
</ol>

Die Autoren erklären, keine widerstreitenden Interessen im Zusammenhang mit dieser Studie.

Das beschriebene Verfahren zur Cholesterin-Efflux gemessen wurde entwickelt, um die Bewegung von Cholesterin aus den Zellen zu einer extrazellulären Cholesterin-Akzeptor zu messen. Es gibt einige kritische Überlegungen zum Verständnis dieser Methode. Kennzeichnung und Äquilibrierung In der beschriebenen Methode markierten Cholesterin im Serum enthaltenden Serum zugegeben. Obwohl dies nicht im Detail untersucht, wird angenommen, dass Cholesterin in Serum-Lipoprot...

<table border="1"><tbody><tr><td> Reagenzien \ Cells </td><td> HeLa </td><td> THP-1 </td><td> RAW </td><td> HUVEC </td><td> BHK-21 </td><td> HFF </td></tr><tr><td> Komplette Medien </td><td colspan="3"> RPMI - 10% FCS - 1% L-Glutamin - 0,2% Pen Strep </td><td> M199 - 5% FCS - 1% L-Glutamin - 1% Pen Strep - 2% HEPES (1M) - 7% Endothelzellwachstum Ergänzung (ECGS) </td><td colspan="2"> DMEM - 10% FCS - 1% L-Glutamin - 0,2% Pen Strep </td></tr><tr><td> Serum-freie Medien </td><td colspan="3"> RPMI - 1% L-Glutamin - 0,2% Pen Strep </td><td> M199 - 1% L-Glutamin - 1% Pen Strep - 2% HEPES (1M) - 7% Endothelzellen wachsenth Ergänzung (ECGS) </td><td colspan="2"> DMEM - 1% L-Glutamin - 0,2% Pen Strep </td></tr><tr><td> Passage-Methode </td><td> 1 x Trypsin </td><td> Aufhängung </td><td> Schaben </td><td colspan="3"> 1 x Trypsin </td></tr><tr><td> Durchtrittsverhältnis </td><td> 03.10 </td><td> 01.03 </td><td></td><td> 01.20 </td><td> 01.03 </td><td> 01.03 </td></tr><tr><td> Zell-Aktivierung (optional) </td><td></td><td colspan="5"> TO-901.317 - LXR-Agonisten - Final konz. 4 uM </td></tr><tr><td> Andere Reagenzien </td><td></td><td> Phorbol-12-myristat-13-acetat (PMA) - Zelldifferenzierung - Final konz. 0.1μg/ml </td><td></td><td></td><td></td><td></td></tr></tbody></table>

cholesterol efflux assay

Cholesteringehalt von Zellen innerhalb der sehr engen Grenzen gehalten werden, zu viel oder zu wenig Cholesterin in einer Zelle führt zu Störungen von Zellmembranen, Apoptose und Nekrose 1. Die Zellen können Quelle Cholesterin aus intrazellulären Synthese und von Plasma-Lipoproteine ​​sind beide Quellen ausreichend, um Zellen vollständig erfüllen die Anforderungen für Cholesterin. Die Prozesse der Cholesterin-Synthese und die Aufnahme ist streng reguliert und Mängel an Cholesterin sind selten zwei. Übermäßige Cholesterin ist häufiger Problem 3. Mit Ausnahme von Hepatozyten und in gewissem Maße NNR-Zellen, Zellen sind nicht in der Lage, Cholesterin zu degradieren. Die Zellen haben zwei Möglichkeiten, um ihre Cholesterin-Gehalt zu reduzieren: auf Cholesterin in Cholesterylester, eine Option mit begrenzten Kapazitäten zu konvertieren, wie eine Überlastung Zellen mit Cholesterylestern ist auch giftig, und Cholesterin Efflux, eine Option, mit potenziell unbegrenzte Kapazität. Cholesterin Efflux ist ein specific Prozess, der durch eine Reihe von intrazelluläre, wie ATP-Bindungs-Cassette-Transporter-Proteine ​​A1 (ABCA1) und G1 (ABCG1) und Scavenger-Rezeptor des Typs B1 reguliert wird. Die natürliche Akzeptor von Cholesterin im Plasma ist High Density Lipoprotein (HDL) und Apolipoprotein AI. Die Cholesterin-Efflux Assay wurde entwickelt, um die Geschwindigkeit der Cholesterin-Efflux aus kultivierten Zellen zu quantifizieren. Es misst die Fähigkeit von Zellen, Cholesterin Efflux und / oder die Kapazität des Plasmas Akzeptoren, Cholesterin aus den Zellen freigesetzt akzeptieren zu halten. Der Assay besteht aus den folgenden Schritten. Schritt 1: Markieren zellulärem Cholesterin mit indem markiertem Cholesterin im Serum enthaltenden Medium und Inkubation mit Zellen für 24-48 h. Dieser Schritt kann mit dem Laden der Zellen mit Cholesterin kombiniert werden. Schritt 2: Inkubation der Zellen in einem Serum-freien Medium auf markiertem Cholesterin unter allen intrazellulären Cholesterin-Pools sich ausgleichen lassen. Dieses Stadium kann mit der Aktivierung von zellulären ch kombiniert werdenolesterol Transporter. Schritt 3: Inkubation der Zellen mit der extrazellulären Akzeptor und Quantifizierung der Bewegung des markierten Zellen Cholesterin aus dem Akzeptor. Wenn Cholesterin Vorstufen wurden verwendet, um neu synthetisierte Cholesterin zu kennzeichnen, einen vierten Schritt, Reinigung von Cholesterin, erforderlich sein. Der Test liefert folgende Informationen: (i), wie eine bestimmte Behandlung (eine Mutation, eine Knock-down, eine Überexpression oder eine Behandlung) die Kapazität der Zelle wirkt sich auf Efflux Cholesterin und (ii), wie die Kapazität des Plasma-Cholesterin-Akzeptoren zu akzeptieren durch eine Krankheit oder eine Behandlung beeinflusst. Diese Methode wird häufig in Zusammenhang mit Herz-Kreislauf-Forschung, metabolische und neurodegenerative Erkrankungen, Infektionskrankheiten und reproduktiven Erkrankungen eingesetzt.

Watch this Scientific Journal Video about Cholesterol Efflux Assay at JoVE.com

Cholesterin-Efflux-Assay | Medizin | Video

Cholesterol Efflux Assay

Die Cholesterin-Assay wurde entwickelt, um die Geschwindigkeit der Cholesterin-Efflux aus kultivierten Zellen und die Kapazität des Plasmas Akzeptoren, Cholesterin aus den Zellen freigesetzt akzeptieren zu quantifizieren. Der Test besteht aus Markieren von Zellen mit Cholesterin, Äquilibrierung von Cholesterin unter den intrazellulären Pools und Freisetzung von Cholesterol an einer extrazellulären Akzeptor.

Cholesterin Efflux-Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

Research

Education

JoVE Journal

JoVE Core

Biology

Medicine

Fundamentals

Chemistry of Life

SCIENTISTS IN ACTION

29.8K Aufrufe.  Baker IDI Heart and Diabetes Institute. Die Cholesterin-Assay wurde entwickelt, um die Geschwindigkeit der Cholesterin-Efflux aus kultivierten Zellen und die Kapazität des Plasmas Akzeptoren, Cholesterin aus den Zellen freigesetzt akzeptieren zu quantifizieren. Der Test besteht aus Markieren von Zellen mit Cholesterin, Äquilibrierung von Cholesterin unter den intrazellulären Pools und Freisetzung von Cholesterol an einer extrazellulären Akzeptor.

Serie: Cholesterol Efflux Assay

Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological processes. This subject embraces diverse scientific areas, spanning from physical, biological and bioorganic chemistry to biology and medicine, with applications to the amelioration of quality of life, health and aging. Multidisciplinary skills are required for the full investigation of the many facets of radical processes in the biological environment and chemical knowledge plays a crucial role in unveiling basic processes and mechanisms. We developed a chemical biology approach able to connect free radical chemical reactivity with biological processes, providing information on the mechanistic pathways and products. The core of this approach is the design of biomimetic models to study biomolecule behavior (lipids, nucleic acids and proteins) in aqueous systems, obtaining insights of the reaction pathways as well as building up molecular libraries of the free radical reaction products. This context can be successfully used for biomarker discovery and examples are provided with two classes of compounds: mono-trans isomers of cholesteryl esters, which are synthesized and used as references for detection in human plasma, and purine 5',8-cyclo-2'-deoxyribonucleosides, prepared and used as reference in the protocol for detection of such lesions in DNA samples, after ionizing radiations or obtained from different health conditions.

Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.

Free Radicals in Chemical Biology: ab chemisches Verhalten, um Biomarker Entwicklung

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological ...

Forschung

Chemie

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

Anreicherung von Säugetiergeweben und Xenopus-Oozyten mit Cholesterin

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

Biochemie

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells.

Spotvariations-Fluoreszenz-Korrelationsspektroskopie zur Analyse der molekularen Diffusion an der Plasmamembran lebender Zellen

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

Zellfreie biochemische fluorometrisch enzymatische Assays für Hochdurchsatz-Messung der Lipidperoxidation in High-Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Immunologie und Infektion

Lipid Exchange Assay in Living Cells

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the outer leaflet drives the membrane&#39;s propensity to form lipid rafts. The transient nature of lipid rafts makes it difficult to study in living cells. Therefore, methods that add or remove raft-forming lipids at&#160;the outer leaflet of living cells facilitate studying the characteristics of rafts, such as their effects on membrane proteins. Lipid exchange experiments developed in our lab utilize lipid-loaded cyclodextrins to remove and add exogenous phospholipids to change the lipid constitution of the plasma membrane. Substituting the membrane with a raft or non-raft-forming lipid can aid in studying the effects on transmembrane protein activity. Here, we describe a method for lipid exchange on the outer leaflet of the plasma membrane using lipid-loaded cyclodextrin. We demonstrate the preparation of the exchange media and the subsequent treatment of attached mammalian cells. We also showcase how to measure the efficiency of exchange using HP-TLC. This protocol yields a nearly complete replacement of the outer leaflet with exogenous lipids without altering cellular viability, permitting further experimentation on modified intact plasma membranes.

We describe a method for using cyclodextrin to mediate exchange between lipids of the plasma membrane with exogenous lipids. This technique can be paired with experiments studying transmembrane proteins, which behave differently in lipid raft-like environments than they do in non-raft-like environments.

Lipidaustausch-Assay in lebenden Zellen

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

Differentielle Effekte von lipidsenkenden Medikamenten Modulation Morphologie der Cholesterin-Partikel

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

Medizin

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Hochdurchsatz-Nitrobenzoxadiazole-Label Cholesterin-Efflux-Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Ein Proteoliposom Basierte Efflux-Assay auf Einzelmoleküleigenschaften von Cl Bestimmen<sup&gt; -</sup&gt; Kanäle und Transporter

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Biologie

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Verwenden von Caco-2-Zellen, die Lipidtransport Studie der Darm

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

LDL-Cholesterin-Aufnahme-Assay mit Live Cell Imaging-Analyse mit Zelle Systemüberwachung

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...