Questo lavoro è stato sostenuto dalle concessioni dal National Health and Medical Research Council of Australia (NHMRC) (526.615 e 545.906) e in parte dal Programma OIS del Governo del Victoria. DS è un collega di NHMRC.

1. Preparazione di [3 H]-colesterolo <ol><li> Nella cappa, erogare la quantità di [3 H] colesterolo in una provetta da microcentrifuga da 1,5 ml (0,5 pCi (19 kBq) per pozzetto è richiesto per un saggio tipico). </li><li> Se [3 H]-colesterolo viene sospeso in toluene, asciugare completamente con gas N2 e risospendere con etanolo al 100% ad una concentrazione finale di 1 microCi (37 kBq / pl. Vortex e mescolare bene. </li></ol> 2. Cellule placcatura e all&#39;etichettatura dei prodotti colesterolo cellulare Questo protocollo è stato testato con i seguenti tipi di cellule: monociti umani 4,5 THP-1, i monociti-macrofagi umani 6,7,8, RAW 264.7 macrofagi murini 5,9,10,11, cellule HeLa 12, vena ombelicale umano endoteliale cellule (HUVEC), cellule BHK-21 9, fibroblasti umani e mouse 13,14, cellule di epatocarcinoma umano HepG2 15 e, in forma modificata, da parte delle piastrine &lt;sup&gt; 16. <ol><li> Risospendere le cellule e contarli. Cellule in piastre 12-pozzetti alla densità finale di 0.2x10 6 cellule per pozzetto in 0,9 ml di mezzo completo. Le cellule continuerà a crescere e raggiungerà 0.8x10 6 cellule per pozzetto dal tempo dell&#39;esperimento efflusso. Per le celle che non dividono, come differenziati cellule THP-1, la densità di semina dovrebbe essere 0.8x10 6 cellule per pozzetto. Se si utilizza 6 - o 24-pozzetti, doppio o metà del numero di cellule per pozzetto, rispettivamente. Il numero di pozzi dovrebbe essere sufficiente per le determinazioni quadruplicato per ogni condizione sperimentale. </li><li> Se la differenziazione di cellule THP-1 è richiesto, aggiungere PMA (concentrazione finale 0,1 mg / ml) ai mezzi per 48-72 h. </li><li> Aggiungere una piccola aliquota di supporto nella provetta da microcentrifuga contenente il [3 H] colesterolo. </li><li> Lavare lati del tubo di supporto prima di aggiungere [3 H] colesterolo separato in una aliquota di mezzi completi (100 pl / pozzetto). </li> &lt;li&gt; Aggiungi i supporti contenenti [3 H] colesterolo nei pozzetti con le cellule (volume finale per pozzetto è di 1 ml). Se nessun trattamento di cellule è necessario prima di etichettatura, le cellule possono essere placcato [3 H]-mezzo contenente colesterolo. </li><li> Incubare le cellule per 48 ore in incubatore per colture cellulare (37 ° C, 5% CO 2). </li></ol> 3. Equilibrazione incubazione <ol><li> Dopo 48 ore di incubazione, controllare le cellule al microscopio. Assicurarsi che sono in buona salute e sono a circa l&#39;80% di confluenza. </li><li> Rimuovere i supporti contenenti [3 H] colesterolo. Lavare delicatamente le cellule con PBS. Ripeti 3 volte lavaggio. </li><li> Preparare senza siero media. Se necessario, attivare le cellule aggiungendo agonista LXR (come TO-901317 a 1-4 mMol / L) o cAMP (0,3 mmol / L; per le cellule di origine solo il mouse) al siero free-media. </li><li> Aggiungere 500 microlitri di siero dei media liberi in ciascun pozzetto. </li><li> Incubare per 18 ore in incubatore per colture cellulari (37 ° C, 5% CO 2). </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Efflusso incubazione Colesterolo <ol><li> Dopo incubazione di 18 ore in terreno privo di siero, le cellule controllare sotto microscopio. Assicurarsi che siano sani e confluenti (minimo 80% di confluenza). </li><li> Preparare la soluzione di colesterolo accettori di efflusso in terreno privo di siero. Esempi di accettori comprendono: Apolipoproteina AI (apoA-I) (concentrazione finale 10 pg / ml) Lipoproteine ​​ad alta densità (HDL) (concentrazione finale 20 pg / ml) Ciclodestrina (concentrazione finale 200 pg / ml) Plasma (concentrazione finale 1-2%) </li><li> Lavare delicatamente le cellule con PBS. </li><li> Aggiungere 250 pl di siero mezzi privi con accettori a ciascun pozzetto. </li><li> Lascia una serie di pozzi per determinare gli efflusso (efflusso di siero mezzi di comunicazione liberi con NO accettore) </li><li> Incubare le cellule per 2 ore in incubatore per colture cellulari (37 ° C, 5% CO 2). Durata della incubazione efflusso possono variare da 30 minuti a 8 ore se necessario. </li></ol> 5. Elaborazione Samples <ol><li> Dopo 2 ore di incubazione controllare le cellule al microscopio. </li><li> Raccogliere media in provette da 1,5 ml microcentrifuga. Spin a 14.000 rpm per 1-10 min a temperatura ambiente per rimuovere i detriti cellulari. </li><li> Trasferire 100 pl supporti in 7 ml fiala di scintillazione. Aggiungere 5 ml di Insta-gel Plus (PerkinElmer) e la miscela di vortice. Conservare i campioni rimasti a 4 ° C. </li><li> Posizionare le piastre in freezer per 30 minuti. Aggiungere 500 ul dH 2 O in ciascun pozzetto. Controllare cellule sotto microscopio a garantire che tutte le cellule hanno sollevato dal fondo dei pozzetti. Se ancora aderente, o lasciare le piastre con dH 2 O a 4 ° C durante la notte pozzi o raschiare. </li><li> Una volta che tutte le cellule sono cominciate, pipettare su e giù per rompere grumi di cellule. Trasferire 100 pl in fiale di scintillazione 7 ml. Aggiungere 5 ml di Insta-gel Plus (PerkinElmer) e la miscela di vortice. Conservare le piastre rimangono a 4 ° C. </li><li> Posizionare fiale di scintillazione sul contatore a scintillazione. Configurare contatore di contare per [3 H] indpm di unità. </li></ol> 6. Analizzando i risultati <ol><li> La velocità di efflusso di colesterolo è solitamente espresso in percentuale del colesterolo trasferito da cellule al accettore. La formula viene utilizzata la seguente: </li></ol><img alt="Equazione 1" src="/files/ftp_upload/3810/3810eq1.jpg" /><ol start="2"><li> L&#39;efflusso specifico viene calcolato come differenza tra l&#39;efflusso in presenza o assenza del accettore (vuoto). </li></ol><img alt="Equazione 2" src="/files/ftp_upload/3810/3810eq2.jpg" /> 7. Timeline <img alt="Figura 1" src="/files/ftp_upload/3810/3810timeline.jpg" /> 8. Risultati rappresentativi Un esempio di un risultato di un esperimento efflusso colesterolo è mostrato in fig. 1. In questo esperimento THP-1 monociti umani sono state differenziate in macrofagi e di efflusso del colesterolo ad accettori diverse è statatestato. Efflusso colesterolo per mezzo senza accettori (&quot;bianco&quot;) era 0,79% e questo valore è stato considerato come un non-specifica efflusso ed è stata sottratta da altri valori. Efflusso di colesterolo per la salute umana apoA-I (concentrazione finale 30 ug / ml) è stato 4,75%. Un campione di plasma di riferimento è stato incluso in questo esperimento di monitorare la variabilità inter-sperimentale. Un campione di riferimento può essere utilizzata per la normalizzazione dei dati attraverso un gran numero di esperimenti, ma abbiamo trovato prudente ripetere il dosaggio se la variabilità è elevata. Un plasma del paziente (concentrazione finale 2%) è stato testato prima e dopo la paziente è stata trattata con farmaci. Si è concluso che in questo farmaco paziente ha avuto un impatto negativo sulla capacità di plasma per supportare efflusso colesterolo. Un altro esempio di un risultato dell&#39;esperimento efflusso è mostrato in fig. 2. In questo esperimento i macrofagi RAW 264.7 sono stati attivati ​​o non attivata tramite incubazione per una notte con agonista LXR TO-901317 (concentrazione finale1 mmol / L) e colesterolo efflusso allo stesso campione di plasma umani (2%) è stato testato. Si è concluso che l&#39;attivazione di espressione cellulare di trasportatori ABC con agonista LXR aumenta la capacità delle cellule di rilasciare il colesterolo per accettore extracellulare. <img alt="Figura 1" src="/files/ftp_upload/3810/3810fig1.jpg" /> Figura 1. Efflusso colesterolo da cellule THP-1 ad accettori diverse. Percentuale di efflusso colesterolo (cioè la percentuale di colesterolo marcato spostato da cellule all&#39;accettore specificato) è mostrato dopo sottrazione dei valori vuoti. Media ± SD di determinazioni quadruplicato, * p &lt;0,05. <img alt="Figura 2" src="/files/ftp_upload/3810/3810fig2.jpg" /> Figura 2. Efflusso di colesterolo da RAW 264,7 attivata o disattivata con agonista LXR al plasma umano. Percentuale di efflusso di colesterolo (cioè la percentuale di colesterolo marcato spostato da celle aaccettore specificato) viene mostrato dopo la sottrazione di valori vuoti. Media ± SD di determinazioni quadruplicato, * 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>
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</ol>

Gli autori dichiarano di non conflitto di interessi relativi a questo studio.

Il metodo descritto per misurare il colesterolo efflusso è progettato per misurare il movimento del colesterolo da cellule ad un accettore colesterolo extracellulare. Ci sono diverse considerazioni critiche nella comprensione di questa metodologia. Etichettatura ed equilibratura Nella metodologia descritta colesterolo marcato viene aggiunto siero contenente siero. Sebbene non esaminati in dettaglio, si presume che il colesterolo è incorporato in lipoproteine ​?...

<table border="1"><tbody><tr><td> Reagenti \ Celle </td><td> HeLa </td><td> THP-1 </td><td> RAW </td><td> HUVEC </td><td> BHK-21 </td><td> HFF </td></tr><tr><td> Mezzi completi </td><td colspan="3"> RPMI - 10% FCS - 1% L-glutammina - Strep Pen 0,2% </td><td> M199 - 5% FCS - 1% L-glutammina - Strep penna 1% - HEPES 2% (1M) - 7% di crescita endoteliale supplemento cellulare (ECG) </td><td colspan="2"> DMEM - 10% FCS - 1% L-glutammina - Strep Pen 0,2% </td></tr><tr><td> Siero media gratuiti </td><td colspan="3"> RPMI - 1% L-glutammina - Strep Pen 0,2% </td><td> M199 - 1% L-glutammina - Strep penna 1% - HEPES 2% (1M) - 7% delle cellule endoteliali crescere° supplemento (ECG) </td><td colspan="2"> DMEM - 1% L-glutammina - Strep Pen 0,2% </td></tr><tr><td> Metodo Passage </td><td> 1 x tripsina </td><td> Sospensione </td><td> Raschiare </td><td colspan="3"> 1 x tripsina </td></tr><tr><td> Passage Rapporto </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> Attivazione delle cellule (opzionale) </td><td></td><td colspan="5"> TO-901317 - LXR-agonisti - Conc finale. 4 pM </td></tr><tr><td> Altri reagenti </td><td></td><td> forbolo 12-miristato 13-acetato (PMA) - La differenziazione delle cellule - Conc finale. 0.1μg/ml </td><td></td><td></td><td></td><td></td></tr></tbody></table>

cholesterol efflux assay

Contenuto di colesterolo delle cellule deve essere mantenuta entro i limiti molto stretti, troppo o troppo poco colesterolo in un risultato cellulari in rottura delle membrane cellulari, l&#39;apoptosi e necrosi 1. Le cellule possono colesterolo di origine da sintesi intracellulare e da lipoproteine ​​plasmatiche, entrambe le fonti sono sufficienti per soddisfare a pieno le esigenze cellule &#39;per il colesterolo. I processi di sintesi del colesterolo e l&#39;assorbimento sono strettamente regolati e carenze di colesterolo sono rare 2. Eccesso di colesterolo è di 3 problema più comune. Con l&#39;eccezione di epatociti e ad alcune celle di laurea corticosurrenali, le cellule sono in grado di degradare il colesterolo. Le cellule hanno due opzioni per ridurre il loro contenuto di colesterolo: il colesterolo per convertire in esteri del colesterolo, un&#39;opzione con una capacità limitata di sovraccaricare le celle con esteri del colesterolo è anche tossico, e di efflusso del colesterolo, un&#39;opzione con una capacità potenzialmente illimitata. Efflusso di colesterolo è una specific processo che è regolato da una serie di trasportatori intracellulari, come ATP binding cassette transporter A1 proteine ​​(ABCA1) e G1 (ABCG1) e del recettore scavenger di tipo B1. L&#39;accettore naturale di colesterolo nel plasma è lipoproteine ​​ad alta densità (HDL) e di apolipoproteina AI. Il saggio di efflusso di colesterolo è stato progettato per quantificare la velocità di efflusso di colesterolo dalle cellule in coltura. Si misura la capacità delle cellule di mantenere efflusso colesterolo e / o la capacità di plasma accettori di accettare il colesterolo rilasciato dalle cellule. Il saggio è costituito dai seguenti passaggi. Fase 1: etichettatura colesterolo cellulare con l&#39;aggiunta di colesterolo marcato al siero contenente medio e incubazione con cellule per 24-48 ore. Questo passaggio può essere combinato con caricamento di cellule con colesterolo. Fase 2: incubazione di cellule a terreno privo di siero di colesterolo marcato equilibrare tra tutti i lotti di colesterolo intracellulare. Questa fase può essere combinato con l&#39;attivazione del cellulare cholesterol trasportatori. Fase 3: incubazione delle cellule con accettore extracellulare e quantificazione di movimento di colesterolo marcato da cellule al accettore. Se precursori del colesterolo sono stati utilizzati per etichettare colesterolo neosintetizzata, una quarta fase, purificazione di colesterolo, può essere richiesto. Il dosaggio fornisce le seguenti informazioni: (i) come un particolare trattamento (una mutazione, un knock-down, una sovraespressione o un trattamento), riguarda la capacità della cellula di colesterolo efflusso e (ii) come la capacità di accettori plasma ad accettare colesterolo è affetto da una malattia o un trattamento. Questo metodo viene spesso utilizzato nel contesto della ricerca cardiovascolare, malattie metaboliche e neurodegenerative, malattie infettive e riproduttivi.

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

Analisi dell'efflusso del colesterolo | Medicina | Video

Cholesterol Efflux Assay

Il saggio del colesterolo è progettato per quantificare il tasso di efflusso colesterolo da cellule coltivate e la capacità di plasma accettori di accettare il colesterolo rilasciato dalle cellule. Il saggio è costituito da cellule con l&#39;etichettatura del colesterolo, del colesterolo equilibrio tra le piscine e il rilascio intracellulare del colesterolo ad un accettore extracellulare.

Efflusso test Colesterolo

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 ...

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29.8K Views.  Baker IDI Heart and Diabetes Institute. Il saggio del colesterolo è progettato per quantificare il tasso di efflusso colesterolo da cellule coltivate e la capacità di plasma accettori di accettare il colesterolo rilasciato dalle cellule. Il saggio è costituito da cellule con l'etichettatura del colesterolo, del colesterolo equilibrio tra le piscine e il rilascio intracellulare del colesterolo ad un accettore extracellulare.

Video: 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.

I radicali liberi in Chemical Biology: da comportamento chimico a sviluppo Biomarker

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

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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.

Arricchimento dei tessuti mammiferi e Xenopus Oocytes con colesterolo

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

Biochimica

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.

Spettroscopia di correlazione di fluorescenza a variazione spot per l'analisi della diffusione molecolare sulla membrana plasmatica di cellule viventi

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.

Analisi enzimatica fluorometrica cell-free biochimico per la misura di High-throughput di perossidazione lipidica in lipoproteina ad alta densità

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

Immunologia e infezioni

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.

Saggio di scambio lipidico in cellule viventi

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.

Effetti differenziali di farmaci ipolipemizzanti nella modulazione della morfologia delle particelle di colesterolo

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

Medicina

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.

Analisi di efflusso di High-throughput Nitrobenzoxadiazole-labeled colesterolo

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.

A Proteoliposome-Based Efflux Assay per determinare le proprietà singola molecola di Cl<sup&gt; -</sup&gt; Canali e trasportatori

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 ...

Biologia

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.

Utilizzando cellule Caco-2 allo Studio Lipid Trasporto dall&#39;intestino

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.

Analisi di assorbimento di colesterolo LDL utilizzando Live Cell Imaging analisi con cella di monitoraggio dello stato

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

Efflusso test Colesterolo

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