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

Dette arbejde blev støttet af tilskud fra National Sundhed og Medical Research Council of Australia (NHMRC) (526.615 og 545.906), og dels fra Victorias regering har OIS Program. DS er en fyr på NHMRC.

1. Fremstilling af [3H]-cholesterol <ol><li> I stinkskabet, dispensere den krævede mængde af [3H] cholesterol i et 1,5 ml mikrofugerør (0,5 uCi (19 kBq) per brønd er nødvendig for en typisk assay). </li><li> Hvis [3H]-kolesterol suspenderes i toluen, tørres ned fuldstændigt med N2-gas og resuspender med 100% ethanol til en slutkoncentration på 1 uCi (37 kBq / ul. Vortexes og bland godt. </li></ol> 2. Udplade celler og Mærkning Cellular Kolesterol <p c...

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Den beskrevne fremgangsmåde til måling cholesterol efflux er designet til at måle bevægelsen af ​​cholesterol fra celler til en ekstracellulær cholesterol acceptor. Der er flere kritiske overvejelser i forståelsen af ​​denne metode. Mærkning og ækvilibrering I den beskrevne metode mærket cholesterol sættes til serumholdigt serum. Selv aldrig undersøgt i detaljer, antages det, at cholesterol er inkorporeret i serumlipoproteiner, og de optages af ce...

<table border="1"><tbody><tr><td> Reagenser \ Celler </td><td> HeLa </td><td> THP-1 </td><td> RAW </td><td> HUVEC </td><td> BHK-21 </td><td> HFF </td></tr><tr><td> Komplette medier </td><td colspan="3"> RPMI - 10% FCS - 1% L-glutamin - 0,2% Pen strep </td><td> M199 - 5% FCS - 1% L-glutamin - 1% Pen strep - 2% HEPES (1M) - 7% endotelcellevækst supplement (ECGS) </td><td colspan="2"> DMEM - 10% FCS - 1% L-glutamin - 0,2% Pen strep </td></tr><tr><td> Serumfrie medier </td><td colspan="3"> RPMI - 1% L-glutamin - 0,2% Pen strep </td><td> M199 - 1% L-glutamin - 1% Pen strep - 2% HEPES (1M) - 7% endotelcelle vokseth tillæg (ECGS) </td><td colspan="2"> DMEM - 1% L-glutamin - 0,2% Pen strep </td></tr><tr><td> Passage Metode </td><td> 1 x Trypsin </td><td> Suspension </td><td> Skrab </td><td colspan="3"> 1 x Trypsin </td></tr><tr><td> Passage Ratio </td><td> 3:10 </td><td> 01:03 </td><td></td><td> 1:20 </td><td> 01:03 </td><td> 01:03 </td></tr><tr><td> Cell Activation (valgfrit) </td><td></td><td colspan="5"> TO-901.317 - LXR-agonist - Slutkonc. 4 uM </td></tr><tr><td> Andre reagenser </td><td></td><td> phorbol 12-myristat 13-acetat (PMA) - Celledifferentiering - Slutkonc. 0.1μg/ml </td><td></td><td></td><td></td><td></td></tr></tbody></table>

cholesterol efflux assay

Cholesterolindholdet af celler skal være inden for meget snævre grænser, for meget eller for lidt cholesterol i en celle resulterer i afbrydelse af cellulære membraner, apoptose og nekrose 1. Celler kan kilde kolesterol fra intracellulær syntese og fra plasmalipoproteiner, begge kilder er tilstrækkelige til fuldt ud at tilfredsstille cellernes krav til kolesterol. Processerne for kolesterol syntese og optagelse er stramt reguleret, og mangler af kolesterol er sjældne 2. Overdreven kolesterol er mere almindeligt problem 3. Med undtagelse af hepatocytter og i nogen grad adrenocorticale celler er celler, i stand til at nedbryde cholesterol. Celler har to muligheder for at reducere deres kolesterol indhold: at konvertere kolesterol i cholesterylestere, en mulighed med begrænset kapacitet, som overbelastning celler med cholesterylestrene er også giftigt, og cholesterol efflux, en option med potentielt ubegrænset kapacitet. Cholesterol efflux er specforsk proces, der reguleres af en række intracellulære transportører, såsom ATP-binding kassetten transportør proteiner A1 (ABCA1) og G1 (ABCG1) og scavenger-receptor type B1. Den naturlige acceptor af cholesterol i plasma er high density lipoprotein (HDL) og apolipoprotein AI. Den cholesterol efflux assayet er designet til at kvantificere graden af ​​cholesterol efflux fra dyrkede celler. Den måler kapaciteten af ​​celler til at opretholde cholesterol efflux og / eller kapaciteten af ​​plasma acceptorer til at modtage cholesterol frigivet fra cellerne. Assayet består af følgende trin. Trin 1: mærkning cellulær cholesterol ved tilsætning mærket cholesterol til serum-holdigt medium og inkubering med celler i 24-48 timer. Dette trin kan kombineres med lastning af celler med cholesterol. Trin 2: inkubation af celler i serumfrit medium for at ækvilibrere mærket cholesterol hos alle intracellulære cholesterol pools. Dette trin kan kombineres med aktivering af cellulære lmolesterol transportører. Trin 3: inkubering af celler med ekstracellulær acceptor og mængdebestemmelse af bevægelse af mærket cholesterol fra celler til acceptoren. Hvis cholesterol forstadier blev anvendt til at mærke nyligt syntetiserede cholesterol, et fjerde trin, oprensning af cholesterol, kan være nødvendig. Analysen leverer følgende oplysninger: (i), hvordan en bestemt behandling (en mutation, en knock-down, en overekspression eller en behandling) påvirker effektiviteten af ​​cellen til efflux kolesterol og (ii) hvordan kapaciteten af ​​plasma acceptorer at acceptere kolesterol er påvirket af en sygdom eller en behandling. Denne metode anvendes ofte i forbindelse med hjerte-kar-forskning, metaboliske og neurodegenerative sygdomme, infektionssygdomme og reproduktive sygdomme.

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

Cholesterol Efflux Assay

Den cholesterol-assay er designet til at kvantificere graden af ​​cholesterol efflux fra dyrkede celler og kapaciteten af ​​plasma acceptorer til at modtage cholesterol frigivet fra cellerne. Analysen består af mærkning af celler med kolesterol, ligevægt af kolesterol blandt intracellulære pools og frigivelse af kolesterol til et ekstracellulært acceptor.

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 ...

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