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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+in+health+and+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+feedback:+from+Schoenheimer's+bottle+to+Scap's+MELADL.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+cellular+cholesterol+accumulation:+basic+concepts+and+physiological+implications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Density+Lipoprotein+Reduces+the+Human+Monocyte+Inflammatory+Response.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Immunodeficiency+Virus+Impairs+Reverse+Cholesterol+Transport+from+Macrophages.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+High-Density+Lipoprotein+Increases+Plasma+High-Density+Lipoprotein+Anti-Inflammatory+Properties+and+Cholesterol+Efflux+Capacity+in+Patients+With+Type+2+Diabetes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+reconstituted+high-density+lipoprotein+leads+to+acute+changes+in+human+atherosclerotic+plaque.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+freezing+on+high-density+lipoprotein+functionality.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure/Function+Relationships+of+Apolipoprotein+A-I+Mimetic+Peptides:+Implications+for+Antiatherogenic+Activities+of+High-Density+Lipoprotein.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cholesteryl+ester+transfer+protein+overexpression+and+inhibition+on+reverse+cholesterol+transport.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+apolipoprotein+A-I-dependent+cholesterol+efflux+elevates+cholesterol+export+from+macrophages+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Different+Regions+of+ATP-Binding+Cassette+Transporter+A1+in+Cholesterol+Efflux.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+A-I+stimulates+the+transport+of+intracellular+cholesterol+to+cell-surface+cholesterol-rich+domains+(caveolae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+harlequin+ichthyosis+delineates+a+key+role+for+abca12+in+lipid+homeostasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Caveolin-1+Enhances+Cholesterol+Efflux+in+Hepatic+Cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+High-Density+Lipoprotein+Attenuates+Platelet+Function+in+Individuals+With+Type+2+Diabetes+Mellitus+by+Promoting+Cholesterol+Efflux.">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>

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 ...

Research

Education

JoVE Journal

JoVE Core

Biology

Medicine

Fundamentals

Chemistry of Life

SCIENTISTS IN ACTION

29.7K 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

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Metastasi di analisi sperimentale

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Ricerca

Medicina

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

In Vivo saggio canina funzione muscolare

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

Un modello murino del saggio Cornea Pocket Study Angiogenesi

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Saggio sferoide Misura TGF-β-indotta Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

Un<em&gt; In vivo</em&gt; Test per la verifica della nave Permeabilità Sangue

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Gli<em&gt; In ovo</em&gt; CAM-saggio come Xenograftmodel per Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Simulazione pancreatica neuroplasticità:<em&gt; In Vitro</em&gt; Dual-neurone Plasticità Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensionale (3D) Tumore Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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 ...

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.

Efflusso test Colesterolo

Riepilogo

Esplora Altri Video

Capitoli in questo video