कोलेस्ट्रॉल तपका परख
Summary
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.
Abstract
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.
Protocol
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| ReagentsCells | HeLa | THP-1 | RAW | HUVEC | BHK-21 | HFF |
| Complete Media | RPMI – 10% FCS – 1% L-glutamine – 0.2% Pen strep |
M199 – 5% FCS – 1% L-glutamine – 1% Pen strep – 2% HEPES (1M) – 7% Endothelial cell growth supplement (ECGS) |
DMEM – 10% FCS – 1% L-glutamine – 0.2% Pen strep |
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| Serum Free Media | RPMI – 1% L-glutamine – 0.2% Pen strep |
M199 – 1% L-glutamine – 1% Pen strep – 2% HEPES (1M) – 7% Endothelial cell growth supplement (ECGS) |
DMEM – 1% L-glutamine – 0.2% Pen strep |
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| Passage Method | 1 x Trypsin | Suspension | Scrape | 1 x Trypsin | ||
| Passage Ratio | 3:10 | 1:3 | 1:20 | 1:3 | 1:3 | |
| Cell Activation (optional) | TO-901317 – LXR-agonist – Final conc. 4 μM |
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| Other Reagents | phorbol 12-myristate 13-acetate (PMA) – Cell differentiation – Final conc. 0.1μg/ml |
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