Name: enrichment of mammalian tissues and xenopus oocytes with cholesterol
Uploaded: 2020-03-25T13:00:00
Description: Watch this Scientific Journal Video about Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol at JoVE.com

Dette arbejde blev støttet af en Scientist Development Grant (11SDG5190025) fra American Heart Association (til A.R.-D.), og af National Institute of Health R01 tilskud AA-023764 (til A.N.B.), og HL-104631 og R37 AA-11560 (til A.M.D).

Alle eksperimentelle procedurer med dyr blev udført på University of Tennessee Health Science Center (UTHSC). Pasning af dyr og forsøgsprotokoller blev gennemgået og godkendt af Animal Care and Use Committee of the UTHSC, som er en institution, der er akkrediteret af Association for Assessment and Accreditation of Laboratory Animal Care International.
1. Berigelse af væv og celler ved hjælp af methyl-β-cyclodextrin mættet med kolesterol
BEMÆRK: Nedenstående ...

Metoder til berigelse af pattedyrvæv og -celler og Xenopus oocytter med kolesterol udgør et effektivt redskab til at undersøge virkningen af forhøjede kolesterolniveauer på individuelle molekylære arter, på komplekse makromolekylære systemer (f.eks. proteiner) og på celle- og organfunktion. I dette dokument har vi beskrevet to komplementære tilgange, der letter sådanne undersøgelser. Først beskrev vi, hvordan man beriger væv og celler med kolesterol ved hjælp af MβCD mættet med kolesterol. Vi vi...

Kolesterol, en stor cellulære lipid, spiller mange kritiske funktionelle og strukturelle roller1,2,,3,,4,,5,,6,,7,8,9. Fra regulering af plasmamembranens fysiske egenskaber til sikring af cellernes levedygtighed, vækst, spredning og tjener som et signal- og forløbermolekyle i et væld af biokemiske veje, er kolesterol en nødvendig komponent, der er nødvendig for normal celle- og organfunktion. Som et resultat, kolesterolmangel resulterer i alvorlige fysiske misdannelser og en række lidelser. På den anden side, selv en lille stigning i kolesterol over fysiologiske niveauer (2-3x) er cytotoksisk1,,2,,10 og har været forbundet med udviklingen af lidelser, herunder hjerte-kar-1111,12,13 og neurodegenerative sygdomme14,15,16,17. Således, at afhøre de kritiske funktioner af kolesterol og til at bestemme effekten af ændringer i kolesterolniveauer, forskellige tilgange, der ændrer indholdet af kolesterol i væv, celler, og Xenopus oocytter er blevet udviklet.
Ændring af kolesterolniveauer i pattedyrvæv og -celler Flere tilgange kan udnyttes til at nedsætte niveauet af kolesterol i væv og celler18. En tilgang indebærer deres eksponering for statiner opløst i lipoprotein-mangelfuld serum til at hæmme HMG-CoA reduktase, som styrer antallet af kolesterol syntese19,20. Men, disse kolesterolsænkende lægemidler hæmmer også dannelsen af ikke-sterol produkter langs mevalonat e pathway. Derfor tilsættes en lille mængde mevalonat for at gøre det muligt at danne disse produkter21 og forbedre specificiteten af denne fremgangsmåde. En anden tilgang til faldende kolesterolniveauer indebærer brug af β-cyclodextrins. Disse glucopyranose monomerer besidder en intern hydrofob hulrum med en diameter, der matcher størrelsen af steroler22, som letter udvinding af kolesterol fra celler, og dermed nedbryder dem fra deres oprindelige kolesterolindhold23. Et eksempel er 2-hydroxypropyl-β-cyclodextrin (HPβCD), et præklinisk lægemiddel, der i øjeblikket testes til behandling af Niemann-Pick type C-sygdommen, en genetisk arvelig dødelig metabolisk lidelse karakteriseret ved lysosomal kolesterolopbevaring24. Niveauet af kolesterol udtynding afhænger af den specifikke afledte anvendes. 25,26,27,28,29HPβCD udtrækker24,f.eks.,30 Især, dog, β-cyclodextrins kan også udtrække andre hydrofobe molekyler ud over kolesterol, som derefter kan resultere i uspecifikke virkninger31. I modsætning til udtynding, celler og væv kan specifikt beriget med kolesterol gennem behandling med β-cyclodextrin, der er blevet præmættet med kolesterol23. Denne fremgangsmåde kan også anvendes som en kontrol for specificiteten af β-cyclodextrin, der anvendes til kolesterol udtynding31. Udtømning af kolesterol fra væv og celler er ligetil og kan opnås ved at udsætte cellerne i 30-60 min til 5 mM MβCD opløst i det medium, der anvendes til opbevaring af cellerne. Denne fremgangsmåde kan resultere i en 50% fald i kolesterolindholdet (f.eks i hippocampal neuroner32, rotte cerebral arterier33). På den anden side, forberedelse af β-cyclodextrin-kolesterol kompleks for kolesterol berigelse af væv og celler er mere kompleks, og vil blive beskrevet i protokollen afsnit.
En alternativ tilgang til berigelse af væv og celler ved hjælp af β-cyclodextrin mættet med kolesterol indebærer brug af LDL, som er afhængig af LDL-receptorer udtrykt i væv / celler18. Mens denne tilgang giver den fordel, at bruge den naturlige kolesterol homøostase maskiner i cellen, det har flere begrænsninger. For det første kan væv og celler, der ikke udtrykker LDL-receptoren, ikke beriges ved hjælp af denne fremgangsmåde. For det andet, LDL partikler indeholder andre lipider ud over kolesterol. Ldl består specifikt af proteinet ApoB100 (25%) og følgende lipider (75%): ~ 6-8% kolesterol, ~ 45-50% cholesteryl ester, ~ 18-24% fosfolipider, og ~ 4-8% triacylglyceroler34. Således levering af kolesterol via LDL partikler er uspecifik. For det tredje, den procentdel af stigningen i kolesterol indhold af LDL i væv og celler, der udtrykker LDL receptor kan være betydeligt lavere end stigningen observeret ved hjælp af cyclodextrin mættet med kolesterol. For eksempel, i en tidligere undersøgelse, berigelse af gnaver cerebral arterier med kolesterol via LDL resulterede i kun en 10-15% stigning i kolesterolniveauer35. I modsætning hertil resulterede berigelse af disse arterier med cyclodextrin mættet med kolesterol som beskrevet i protokolafsnittet i >50% stigning i kolesterolindholdet (se afsnittet Repræsentative resultater, figur 1).
Ændring af kolesterolniveauer i Xenopus oocytter Xenopus oocytter udgør et heterologt ekspressionssystem, der almindeligvis anvendes til at studere celle- og proteinfunktion. Tidligere undersøgelser har vist, at kolesterol til fosfolipid molære forholdet i Xenopus oocytter er 0,5 ± 0,136. På grund af denne iboende højt niveau af kolesterol, øge indholdet af kolesterol i dette system er udfordrende, men kan opnås ved hjælp af dispersioner fremstillet af membran fosfolipider og kolesterol. De fosfolipider, som vi har valgt til dette formål, svarer til dem, der anvendes til dannelse af kunstige planar lipid bilag og omfatter L-α-phosphatidylethanolamin (POPE) og 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serin (POPS), som beskrevet i afsnittet protokol. Denne fremgangsmåde kan resultere i >50% stigning i kolesterolindholdet (se afsnittet Repræsentative resultater, figur 2).
En alternativ tilgang til berigelse af Xenopus oocytter med fosfolipid-baserede dispersioner indebærer brug af cyclodextrin mættet med kolesterol, hvilket svarer til den måde væv og celler er beriget. Men vi har fundet denne tilgang til at være af lav reproducerbarhed og effektivitet, med et gennemsnit på ~ 25% stigning i kolesterolindholdet. Dette skyldes muligvis disse to tilganges forskellige lasteevne (se afsnittet repræsentative resultater, figur 3). I modsætning hertil har det vist sig, at ved hjælp af cyclodextrin at nedbryder kolesterol fra Xenopus oocytter kan resultere i en ~ 40% fald i kolesterolindhold36.
Her fokuserer vi på kolesterol berigelse af pattedyr væv og celler gennem anvendelse af cyclodextrin mættet med kolesterol, og af Xenopus oocytter ved hjælp af liposomer. Begge tilgange kan udnyttes til at afgrænse effekten af forhøjede niveauer af kolesterol på protein funktion. Mekanismerne for kolesterolmodulering af proteinfunktion kan omfatte direkte interaktioner8 og/eller indirekte virkninger9. Når kolesterol påvirker protein funktion via direkte interaktioner, effekten af en stigning i kolesterolniveauer på protein aktivitet er sandsynligvis uafhængig af celletype, ekspressionssystem, eller berigelse tilgang. For eksempel, Vi udnyttede disse to tilgange til at bestemme effekten af kolesterol på G-protein gated indadrettekalium (GIRK) kanaler udtrykt i atriemyocytter37, hippocampal neuroner32,38, HEK29339 celler, og Xenopus oocytter32,37. Resultaterne af disse undersøgelser var konsistente: i alle tre typer pattedyrceller og i padderoocytter kolesterol upregulated GIRK kanalfunktion (se repræsentant resultater afsnit, Figur 4, for hippocampal neuroner og de tilsvarende eksperimenter i Xenopus oocytter). Endvidere, observationerne i disse undersøgelser var også i overensstemmelse med resultaterne af undersøgelser udført i atriemyocytter37,40 og hippocampal neuroner32,38 frisk isoleret fra dyr udsat for en høj kolesterol kost40. Især kolesterol berigelse af hippocampal neuroner ved hjælp af MβCD vendt effekten af atorvastatin terapi anvendes til at løse virkningen af højt kolesteroltal kost både på kolesteroltallet og GIRK funktion38. I andre undersøgelser undersøgte vi effekten af mutationer på kolesterolfølsomheden af den indadvendte korrigere kaliumkanal Kir2.1 ved hjælp af både Xenopus oocytter og HEK293 celler41. Igen var mutationernes virkning på kanalens følsomhed ens i de to systemer.
Anvendelserne af begge berigelse metoder til bestemmelse af virkningen af forhøjede kolesterolniveauer på molekylære, cellulære, og organfunktion er talrige. Især er brugen af cyclodextrin-kolesterol komplekser til at berige celler og væv meget almindeligt i vid udstrækning på grund af dens specificitet. Nylige eksempler på denne tilgang omfatter bestemmelse af virkningen af kolesterol på HERG kanal aktivering og underliggende mekanismer42, opdagelsen af, at kolesterol aktiverer G-protein koblet receptor glattet til at fremme Pindsvin signalering43, og identifikation af den rolle, kolesterol i stamceller biomekanik og adipogenesis gennem membran-associerede linker proteiner44. I vores eget arbejde, vi udnyttede pattedyr væv berigelse med MβCD: kolesterol kompleks til at studere effekten af kolesterol berigelse på grundlæggende funktion og farmakologisk profil af calcium- og spænding-gated kanaler af stor ledningsevne (BK, MaxiK) i vaskulære glatte muskel35,45,46. I andre undersøgelser, Vi brugte fosfolipid-baserede dispersion tilgang til at berige Xenopus oocytter med kolesterol til at bestemme rollerne af forskellige regioner i Kir2.1 og GIRK kanaler i kolesterol følsomhed41,47,48,49, samt at bestemme formodede kolesterol bindende steder i disse kanaler32,50,51.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amplex Red Cholesterol Assay Kit</td>
			<td>Invitrogen</td>
			<td>A12216</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
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		<tr>
			<td>Pre-Diluted Protein Assay Standards BSA set</td>
			<td>Thermo Scientific</td>
			<td>23208</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain PE 25Mg in Chloroform</td>
			<td>Avanti Lipids</td>
			<td>840022C</td>
			<td></td>
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		<tr>
			<td>16:0-18:1 PS 25Mg Chloroform</td>
			<td>Avanti Lipids</td>
			<td>840034C</td>
			<td></td>
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		<tr>
			<td>Cholesterol 100Mg Powder</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td></td>
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		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>P217</td>
			<td></td>
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			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td></td>
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			<td>HEPES</td>
			<td>Corning</td>
			<td>61-034-RO</td>
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			<td>MgCl2</td>
			<td>Fisher</td>
			<td>M33</td>
			<td></td>
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			<td>NaCl</td>
			<td>Fisher</td>
			<td>S271</td>
			<td></td>
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			<td>KH2PO4</td>
			<td>Fisher</td>
			<td>P285</td>
			<td></td>
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		<tr>
			<td>MgSO4</td>
			<td>EMD Chemicals</td>
			<td>MX0070-1</td>
			<td></td>
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			<td>EDTA</td>
			<td>VWR</td>
			<td>E177</td>
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			<td>Dextrose Anhydrous</td>
			<td>Fisher</td>
			<td>BP350</td>
			<td></td>
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			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td></td>
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		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C3881</td>
			<td></td>
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		<tr>
			<td>Blood Gas Tank</td>
			<td>nexAir</td>
			<td></td>
			<td></td>
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			<td>NaOH</td>
			<td>Fisher</td>
			<td>S318</td>
			<td></td>
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			<td>1.5mL tubes</td>
			<td>Fisher</td>
			<td>S35818</td>
			<td></td>
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		<tr>
			<td>Gastight Syringe 100uL</td>
			<td>Hamilton</td>
			<td>1710</td>
			<td></td>
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		<tr>
			<td>Microliter Syringe 25uL</td>
			<td>Hamilton</td>
			<td>702</td>
			<td></td>
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			<td>12 mL heavy duty conical centrifuge beaded rim tube</td>
			<td>Pyrex</td>
			<td>8120-12</td>
			<td></td>
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			<td>Chloroform</td>
			<td>Fisher</td>
			<td>C298</td>
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		<tr>
			<td>Support Stand</td>
			<td>Homescience Tools</td>
			<td>CE-STAN5X8</td>
			<td></td>
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		<tr>
			<td>Universal Clamp, 3-Prong</td>
			<td>Homescience Tools</td>
			<td>CE-CLPUNIV</td>
			<td></td>
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		<tr>
			<td>Sonicator</td>
			<td>Laboratory Supplies</td>
			<td>G112SP1G</td>
			<td></td>
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		<tr>
			<td>3D rotator mixer</td>
			<td>Benchmark Scientific</td>
			<td>B3D 1308</td>
			<td></td>
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		<tr>
			<td>96 well plate</td>
			<td>Sigma</td>
			<td>BR781602</td>
			<td></td>
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		<tr>
			<td>N2 gas</td>
			<td>nexAir</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beakers 40ml-1L</td>
			<td>Fisher</td>
			<td>02-540</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice Machine</td>
			<td>Scotsman</td>
			<td>CU1526MA-1</td>
			<td></td>
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		<tr>
			<td>Ice bucket</td>
			<td>Fisher</td>
			<td>50-136-7764</td>
			<td></td>
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		<tr>
			<td>1X PBS</td>
			<td>Corning</td>
			<td>21-031-CM</td>
			<td></td>
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		<tr>
			<td>TritonX</td>
			<td>Fisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic Dismembrator</td>
			<td>Fisher</td>
			<td>Model 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microcentrifuge</td>
			<td>Eppendorf</td>
			<td>Model 5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber bottles</td>
			<td>Fisher</td>
			<td>03-251-420</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#8482; Disposable Glass Pasteur Pipets</td>
			<td>FIsher</td>
			<td>13-678-4A</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>FIsher</td>
			<td>50-998-944</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp&#8482; BOD Refrigerated Incubator</td>
			<td>FIsher</td>
			<td>97-990E</td>
			<td></td>
		</tr>
		<tr>
			<td>Oocytes</td>
			<td>Xenoocyte&#8482;</td>
			<td>10005</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>Envigo</td>
			<td>Sprague Dawley</td>
			<td>weight 250g</td>
		</tr>
		<tr>
			<td>Methyl-&#946;-cyclodextrin</td>
			<td>Sigma</td>
			<td>C4555</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath incubator with shaker</td>
			<td>Precision</td>
			<td>51221080</td>
			<td>Lowest shaker setting O/N 37 &#176;C</td>
		</tr>
		<tr>
			<td>Filipin</td>
			<td>Sigma</td>
			<td>SAE0088-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher</td>
			<td>BP231</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 4%</td>
			<td>Mallinckrodt</td>
			<td>2621</td>
			<td></td>
		</tr>
		<tr>
			<td>DI H2O</td>
			<td>University DI source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagnet</td>
			<td>Invitrogen</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Microslides 75x25mm Frosted</td>
			<td>Diagger</td>
			<td>G15978A</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Coverslip</td>
			<td>Diagger</td>
			<td>G15972B</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>Revlon</td>
			<td>771 Clear</td>
			<td></td>
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		<tr>
			<td>Labeling Tape</td>
			<td>Fisher</td>
			<td>15-901-20F</td>
			<td></td>
		</tr>
		<tr>
			<td>Securline Lab Marker II</td>
			<td>Sigma</td>
			<td>Z648205-5EA</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 10mL Syringe</td>
			<td>Fisher</td>
			<td>14-823-16E</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2 &#956;m syringe filter</td>
			<td>VWR</td>
			<td>28150-958</td>
			<td></td>
		</tr>
		<tr>
			<td>KimWipes</td>
			<td>Fisher</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr>
			<td>pH probe</td>
			<td>Sartorus</td>
			<td>py-p112s</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Denver instrument</td>
			<td>Model 225</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ETOH</td>
			<td>Pharmco</td>
			<td>211USP/NF</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Fisher</td>
			<td>02-261-840</td>
			<td></td>
		</tr>
		<tr>
			<td>Steno book</td>
			<td>Staples</td>
			<td>163485</td>
			<td></td>
		</tr>
	</tbody>
</table>

enrichment of mammalian tissues and xenopus oocytes with cholesterol

Kolesterol berigelse af pattedyr væv og celler, herunder Xenopus oocytter anvendes til at studere cellefunktion, kan opnås ved hjælp af en række forskellige metoder. Her beskriver vi to vigtige tilgange, der anvendes til dette formål. Først beskriver vi, hvordan man beriger væv og celler med kolesterol ved hjælp af cyclodextrin mættet med kolesterol ved hjælp af cerebrale arterier (væv) og hippocampale neuroner (celler) som eksempler. Denne fremgangsmåde kan bruges til enhver type væv, celler eller cellelinjer. En alternativ tilgang til kolesterol berigelse indebærer brug af low-density lipoprotein (LDL). Fordelen ved denne fremgangsmåde er, at det bruger en del af den naturlige kolesterol homøostase maskiner i cellen. Mens cyclodextrin-metoden kan anvendes til at berige enhver celletype af interesse med kolesterol, er LDL-metoden dog begrænset til celler, der udtrykker LDL-receptorer (f.eks. leverceller, knoglemarvsafledte celler såsom blodleukocytter og vævsmakrofager), og graden af berigelse afhænger af koncentrationen og mobiliteten af LDL-receptoren. Endvidere, LDL partikler omfatter andre lipider, så kolesterol levering er uspecifik. For det andet, Vi beskriver, hvordan man berigeR Xenopus oocytter med kolesterol ved hjælp af en fosfolipid-baseret dispersion (dvs. liposomer), der omfatter kolesterol. Xenopus oocytter udgør et populært heterologt udtrykssystem, der anvendes til at studere celle- og proteinfunktion. For både cyclodextrin-baserede kolesterol berigelse tilgang pattedyr væv (cerebral arterier) og for fosfolipid-baserede kolesterol berigelse tilgang af Xenopus oocytter, viser vi, at kolesterolniveauer nå et maksimum efter 5 min inkubation. Dette niveau af kolesterol forbliver konstant i længere perioder med inkubation (f.eks. 60 min). Tilsammen danner disse data grundlag for optimerede tidsmæssige betingelser for kolesterolberigelse af væv, celler og Xenopus oocytter til funktionelle undersøgelser, der har til formål at afhøre virkningen af kolesterolberigelse.

Brugen af cyclodextrin mættet med kolesterol som et middel til at berige væv og celler med kolesterol er veletableret. Her viser vi først anvendelsen af denne udbredte tilgang til berigelse af rottecerebrale arterier med kolesterol ved hjælp af MβCD mættet med kolesterol. Figur 1A viser et eksempel på et afbildet cerebralarterieglat muskellag og viser den koncentrationsafhængige stigning i filippineret fluorescens opnået ved vævsber...

Watch this Scientific Journal Video about Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol at JoVE.com

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

To metoder til kolesterol berigelse præsenteres: anvendelse af cyclodextrin mættet med kolesterol til at berige pattedyr væv og celler, og brugen af kolesterol-beriget fosfolipid-baserede dispersioner (liposomer) til at berige Xenopus oocytter. Disse metoder er medvirkende til at bestemme virkningen af forhøjede kolesterolniveauer i molekylære, cellulære, og organfunktion.

Berigelse af pattedyrvæv og Xenopus Oocytter med kolesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

Forhøjet kolesterol er en væsentlig risikofaktor for hjerte-kar-og neurodegenerative sygdom. Vores protokoller giver værdifulde værktøjer til at studere både fysiologiske og mekanistiske konsekvenser af hyperkolesterolæmi. Disse procedurer kan udføres ved hjælp af grundlæggende lab udstyr, og gælder for celler, væv, og Xenopus oocytter.Kolesterol er en vigtig komponent i cellulære membraner i hele kroppen. Disse teknikker kan udnyttes til at studere virkningen af forhøjede niveauer af kolesterol i enhver celletype. Dissekere arterierne er det sværeste skridt.Fjern forsigtigt arterien fra hjernen, uden at strække eller beskadige det vaskulære væv. Lipider er meget delikat, og at arbejde med dem er mere af en kunst end en videnskab. Video demonstration giver en guide til at lære at håndtere lipider omhyggeligt.For at forberede kolesterolmættet methyl-beta-cyclodextrin opløsning, først tilsættes 064 gram methyl-beta-cyclodextrin til 10 milliliter PBS, omrøring opløsningen med en røre bar, for at sikre, at methyl-beta-cyclodextrin fuldt opløses. Dernæst tilsættes 0024 gram kolesterol pulver til kolben, og rør opløsningen kraftigt, ved hjælp af en spatel til at bryde op så mange kolesterol stykker som muligt. Når næsten alle kolesterol er blevet opløst, forsegle kolben med mindst to lag paraffin film.Og ryst kolben på omkring 30 svingninger i minuttet i en 37 grader Celsius vandbad natten over. Efter otte til 16 timer afkøles opløsningen til stuetemperatur, før blandingen filtreres gennem et 22 mikrometer polyethersulfon sprøjtefilter i en klasseflaske. For pattedyr cerebral arterie berigelse, høste hjernen fra en 250 til 300 gram Sprague Dawley rotte i et bæger af PBS på is, før overføre hjernen til en voksbehandlet dissektion skål, under en diserning mikroskop, der indeholder lige nok PBS at nedsænke vævet.Fastgør hjernen med to til tre ben, og sørg for at de ikke trænger blodkarrene. Og bruge skarpe kraftbånd og små kirurgiske saks til forsigtigt dissekere cerebral arterier og deres grene, der danner Circle of Willis i bunden af hjernen. Dernæst skylles kortvarigt op til en centimeter lang arteriesegmenter i PBS, før de skyllede segmenter inkuberes i nok kolesterolberigende opløsning til at dække vævene.For at bestemme eventuelle ændringer i kolesteroltallet, bruge en frisk flaske filipin pulver til at forberede en 10 milligram per milliliter bestand opløsning af farvestoffet i dimethylsulfoxid, og vask kolesterol beriget væv med tre fem minutters vasker i frisk PBS. Efter den sidste vask, fastgør arteriesegmenterne i 4% paraformaldehyd i 15 minutter på is, beskyttet mod lys, før permeabilizing prøverne i 5% Triton i PBS i 10 minutter ved stuetemperatur. Ved inkubationens afslutning vaskes vævet med tre fem minutters vask i PBS på en shaker, inden prøverne tilsættes til en slutkoncentration på 25 mikrogram pr. milliliter af filipinfarveopløsning i en time ved stuetemperatur, beskyttet mod lys.Ved slutningen af inkubationen vaskes arteriestykkerne tre gange, efterfulgt af en kort skylning med destilleret vand. Efter den sidste vask, bruge en lab væv til at absorbere overskydende væske, og montere arterierne på et dias ved hjælp af en passende montering medium. Omhyggeligt dække arterierne med et dæksel slip, idet du skal passe på at undgå rullende eller vridning af vævet, og lad dias tørre i 24 timer ved stuetemperatur, beskyttet mod lys.Når monteringsmediet er tørt, forsegles dækslets længdekanter med klar neglelak, og poleren tørres i 10 til 15 minutter. Derefter billede vævet med en excitation af 340 til 380 nanometer, og en emission på 385 til 470 nanometer. For at forberede fosfolipid baseret dispersion, kombinere 200 mikroliter af 10 milligram per milliliter chloroform opløst lipid opløsning, L-alpha-phosphatidylethanolamin, 1-Palmitoyl-2-oleoyl-sn-glycero-3-fosforcholin, og kolesterol i en 12 milliliter glasrør.Fordampechloroformen i emhætten under en nitrogenstrøm, før lipiderne suspenderes i 800 mikroliter af bufferet 150 millimolar kaliumchlorid og 10 millimolar Tris HEPES opløsning. Derefter forsegle røret med paraffin film, og forsigtigt sonikere rørindholdet på 80 kilohertz i 10 minutter, indtil en mælkeagtig blanding er dannet. For kolesterol berigelse af Xenopus oocytter, bruge skarpe kraftbefæstelser til at forstyrre æggestokkene sæk fra en kvindelig Xenopus laevis frøen i flere regioner, og placere æggestokkene stykker i en 60 millimeter plade med fem milliliter calciumfri ND96 suppleret med 5 milligram pr. milliliter kollagenase.Ryst vævet på en orbital shaker ved 60 svingninger i minuttet i 15 minutter ved stuetemperatur. Ved slutningen af inkubationen skal du bruge en overførselspipette med en bred spids til kraftigt at pipette den oocytholdige opløsning fem til 10 gange for at isolere de enkelte oocytter og skyl hurtigt den mørke oocytopløsning med frisk calciumfri ND96, indtil opløsningen bliver gennemsigtig. Dernæst overføre 90 mikroliter af kolesterol beriget fosfolipid baseret dispersion i en brønd af en 96 brønd plade, og tilføje op til seks oocytter til brønden med så lidt medium som muligt.Placer 96 godt plade på en tredimensionel platform rotator i fem til 10 minutter. Derefter tilføje en dråbe ND96 til brønden, og overføre kolesterol beriget oocytter til en 35 millimeter plade, der indeholder ND96 for deres umiddelbare analyse. Her, et eksempel på en afbildet cerebral arterie glat muskel lag viser koncentrationen afhængige stigning i filipin associeret fluorescens opnået ved væv berigelse med stigende koncentrationer af kolesterol.Især tre timer efter behandlingen med methyl-beta-cyclodextrin kolesterol kompleks kolesterol niveauer faldt med ca 50% i forhold til deres niveau umiddelbart efter berigelse. Mens en time inkubationstid er almindeligt anvendt til at berige væv og celler med kolesterol i løbet af denne tilgang, fem minutters inkubation er normalt tilstrækkelig til at opnå en statistisk signifikant stigning i cerebral arteriel kolesterol indhold som bestemt af kolesterol oxidase baseret biokemisk analyse. Mens ingen væsentlig ændring er observeret i kolesterolniveauer i Xenopus oocytter beriget med kontrol fosfolipid baseret dispersioner mangler kolesterol, kolesterolniveauer stige betydeligt efter kun fem minutters behandling med fosfolipid baseret dispersioner, der omfatter kolesterol, og forblive på dette niveau, når inkubationstiden øges til 60 minutter.Effektiviteten af den cyclodextrinbaserede tilgang til berigende celler er også påvist i neuroner, der er frisk isoleret fra HIPPOcampus ca. Faktisk, inkubation af neuroner i methyl-beta-cyclodextrin mættet med kolesterol i 60 minutter resulterer i en over to gange stigning i kolesteroltallet, vurderet af filipin tilhørende fluorescens. Samlet set er de to vigtigste skridt for disse procedurer forbereder kolesterol berigelse blandingen og sikre integriteten af arteriel væv.Efter kolesterol berigelse, kan man studere funktionen af de proteiner, der er påvirket af hyperkolesterolæmi. Evnen til at berige celler med kolesterol lettet studiet af forhøjede kolesterolniveauer i kardiomyocytter og neuroner. Brugen af oocytter tillod os at undersøge, hvordan kolesterol binder sig til ionkanaler.En laboratoriekittel og handsker hun altid være slidt for denne protokol. Chloroform og paraformaldehyd skal anvendes under emhætte og kasseres som farligt affald.

enrichment-of-mammalian-tissues-and-xenopus-oocytes-with-cholesterol

Research

JoVE Journal

Biochemistry

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Protein Complex Affinity Capture fra Cryomilled pattedyrsceller

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Spændingsklemme Fluorometri i<em&gt; Xenopus</em&gt; Oocytter ved hjælp af fluorescerende unaturlige aminosyrer

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

Berigelse af Detergent-uopløselige Protein aggregater fra Postmortem menneskehjerne

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Imaging Amyloid væv farves med selvlysende konjugeret Oligothiophenes af Hyperspectral Konfokal mikroskopi og fluorescens levetid Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Toeprinting analyse af oversættelse indledningen kompleks dannelse på pattedyr mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

Proteinekspression og -oprensning af pattedyr Bestrophin Ion kanaler

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

Påvisning af proteinubiquitinationssteder ved peptidberigelse og massespektrometri

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

At studere RNA Interactors af Protein Kinase RNA-aktiveret under pattedyr cellecyklus

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

Berigelse af Native lipoprotein partikler med microRNA og efterfølgende bestemmelse af deres absolutte/relative microRNA indhold og deres cellulære overførselshastighed

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

Kvantificering af coenzym A i celler og væv

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...