Name: cholesterol efflux assay
Uploaded: 2012-03-06T13:00:00
Description: Watch this Scientific Journal Video about Cholesterol Efflux Assay at JoVE.com

यह काम राष्ट्रीय स्वास्थ्य और ऑस्ट्रेलिया के चिकित्सा अनुसंधान परिषद (NHMRC) (526,615) और 545,906 से और भाग में विक्टोरियन सरकार OIS प्रोग्राम द्वारा अनुदान द्वारा समर्थित किया गया. डी एस NHMRC के एक साथी है.

1. कोलेस्ट्रॉल [3 घंटे] की तैयारी <ol><li> धूआं हुड में, एक 1.5 मिलीलीटर microfuge ट्यूब (0.5 प्रति μCi (19 kBq) के एक ठेठ परख के लिए आवश्यक है) में 3 [एच] कोलेस्ट्रॉल के लिए आवश्यक राशि वितरित. </li><li> यदि 3 [एच] कोलेस्ट्रॉल ट...

<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. 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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. 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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>

वर्णित विधि कोलेस्ट्रॉल तपका को मापने के लिए एक बाह्य कोलेस्ट्रॉल स्वीकर्ता कोशिकाओं से कोलेस्ट्रॉल के आंदोलन को मापने के लिए डिज़ाइन किया गया है. इस पद्धति को समझने में कई महत्वपूर्ण विचार कर रहे है...

<table border="1"><tbody><tr><td> अभिकर्मकों \ कक्ष </td><td> HeLa </td><td> THP-1 </td><td> रॉ </td><td> HUVEC </td><td> मकान - 21 </td><td> HFF </td></tr><tr><td> पूरा मीडिया </td><td colspan="3"> RPMI - 10% एफसीएस - 1% एल glutamine पेन strep - 0.2% </td><td> M199 - 5% एफसीएस - 1% एल glutamine - 1% पेन strep 2% HEPES (1M -) 7% Endothelial सेल के विकास के पूरक (ECGs) </td><td colspan="2"> DMEM - 10% एफसीएस - 1% एल glutamine पेन strep - 0.2% </td></tr><tr><td> सीरम मुक्त मीडिया </td><td colspan="3"> RPMI - 1% एल glutamine पेन strep - 0.2% </td><td> M199 - 1% एल glutamine - 1% पेन strep 2% HEPES (1M -) 7% Endothelial सेल के विकासवें पूरक (ECGs) </td><td colspan="2"> DMEM - 1% एल glutamine पेन strep - 0.2% </td></tr><tr><td> पारित होने विधि </td><td> 1 एक्स trypsin </td><td> निलंबन </td><td> परिमार्जन </td><td colspan="3"> 1 एक्स trypsin </td></tr><tr><td> पारित होने अनुपात </td><td> 3:10 </td><td> 1:03 </td><td></td><td> 1:20 </td><td> 1:03 </td><td> 1:03 </td></tr><tr><td> सेल सक्रियकरण (वैकल्पिक) </td><td></td><td colspan="5"> - 901,317 LXR-agonist अंतिम सान्द्र. 4 माइक्रोन </td></tr><tr><td> अन्य अभिकर्मकों </td><td></td><td> 12 - myristate phorbol 13-एसीटेट (PMA) सेल भेदभाव अंतिम सान्द्र. 0.1μg/ml </td><td></td><td></td><td></td><td></td></tr></tbody></table>

cholesterol 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 cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

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

Cholesterol Efflux Assay

कोलेस्ट्रॉल परख संवर्धित कोशिकाओं से कोलेस्ट्रॉल तपका की दर और प्लाज्मा स्वीकारकर्ताओं की क्षमता quantitate कोशिकाओं से जारी कोलेस्ट्रॉल को स्वीकार करने के लिए डिज़ाइन किया गया है. परख है intracellular पूल और कोलेस्ट्रॉल की एक बाह्य स्वीकर्ता के लिए जारी बीच कोलेस्ट्रॉल, कोलेस्ट्रॉल का संतुलन के साथ कोशिकाओं लेबलिंग के होते हैं.

कोलेस्ट्रॉल तपका परख

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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