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

Эта работа была поддержана грантами национального здравоохранения и медицинских исследований Совета Австралии (NHMRC) (526615 и 545906) и частично OIS Программа викторианской правительства. DS является членом NHMRC.

1. Подготовка [3 H]-холестерина <ol><li> В вытяжной шкаф, отказаться от требуемого количества [3 H] холестерина в 1,5 мл микроцентрифужную трубки (0,5 мКи (19 кБк) в каждую лунку необходимо для типичного анализа). </li><li> Если [3 H]-холестерина приостановлена ​​в толуоле, высушите его ...

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Описанный метод измерения холестерина истечения предназначен для измерения движения холестерина из клеток внеклеточную акцептором холестерина. Есть несколько важных соображений, в понимании этой методологии. Маркировка и равновесия В описанной методо?...

<table border="1"><tbody><tr><td> Реагенты \ Клетки </td><td> HeLa </td><td> THP-1 </td><td> RAW </td><td> HUVEC </td><td> ВНК-21 </td><td> HFF </td></tr><tr><td> Полное СМИ </td><td colspan="3"> RPMI - 10% FCS - 1% L-глутамина - 0,2% Pen стрептококк </td><td> M199 - 5% FCS - 1% L-глутамина - 1% Pen стрептококк - 2% HEPES (1M) - 7% эндотелиальных дополнение рост клеток (ЭКГ) </td><td colspan="2"> DMEM - 10% FCS - 1% L-глутамина - 0,2% Pen стрептококк </td></tr><tr><td> Сыворотка свободные СМИ </td><td colspan="3"> RPMI - 1% L-глутамина - 0,2% Pen стрептококк </td><td> M199 - 1% L-глутамина - 1% Pen стрептококк - 2% HEPES (1M) - 7% эндотелиальных клеток растиго дополнения (ЭКГ) </td><td colspan="2"> DMEM - 1% L-глутамина - 0,2% Pen стрептококк </td></tr><tr><td> Прохождение метод </td><td> 1 х трипсина </td><td> Приостановление </td><td> Скоблить </td><td colspan="3"> 1 х трипсина </td></tr><tr><td> Прохождение соотношение </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> Сотовые активации (опционально) </td><td></td><td colspan="5"> К-901 317 - LXR-агонистов - Окончательный конц. 4 мкМ </td></tr><tr><td> Другие реагенты </td><td></td><td> форбол 12-миристат-13-ацетат (PMA) - Дифференциация клеток - Окончательный конц. 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

Холестерин анализ предназначен для количественного определения скорости оттока холестерина из культивируемых клеток и способность плазмы акцепторов принять холестерина выпустили из клеток. Анализ состоит из маркировки клеток холестерина, холестерина равновесия между внутриклеточными бассейны и выпуск холестерина внеклеточной акцептором.

Холестерин Анализ истечения

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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