Name: a rapid and specific microplate assay for the determination of intra- and extracellular ascorbate in cultured cells
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Description: Watch this Scientific Journal Video about A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells at JoVE.com

Vi er takknemlige til Dr Stephen Robinson og Ms Hania Czerwinska (Monash University) for den generøse tilbudet av astrocyte kulturer.

En. Bestem Intracellulær Askorbat i dyrkede celler <ol><li> Cellekultur og høsting <ol><li> Grow suspensjon (f.eks menneskelig erythroleukemia, K562) eller heftende celler (f.eks primærastrocytes) ved hjelp av standard kultur prosedyrer 19-21,32,38. Merk: For å sikre at celler inneholder askorbat, legger dyrkede celler med askorbat enten som askorbat eller DHA 33,39. </li></ol></li><li> Lag en askorbat holdige celleekstrakt <ol><li> Inkuber ...

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I denne artikkelen presenterer vi to raske, konkrete og relativt sensitive kolorimetriske mikro analyser for bestemmelse av askorbat avledet fra intra-og ekstracellulære avdelinger i dyrkede celler. Analysene kan gjennomføres med tilgang til standard laboratorieutstyr og reagenser. Den bare moderat kostbart reagens som kreves for analysen er AO, noe som er viktig fordi det gir en høy grad av analytt spesifisitet mot L-askorbat. Prøvene er godt egnet til enten suspensjon celler (f.e...

Oppdagelsen av den kjemiske natur av askorbinsyre (vitamin C), og sin identifikasjon som den etterlengtede &quot;anti-scorbutic faktor&quot;, av Albert Szent-Györgyi og andre i artikler publisert 1928-1934 1 var landemerke hendelser i historien i biokjemi. Faktisk disse funnene bidro til Szent-Györgyi blir tildelt Nobelprisen i fysiologi eller medisin i 1937. Den stadig voksende rekke roller for askorbat i dyre-og plantefysiologi, samt helse, fortsetter å være undersåtter av aktiv vitenskapelig etterforskning og kontrovers. L-askorbat er en rikelig fysiologisk Reduksjon og enzymkofaktor i pattedyr systemer, og bidrar til en rekke veldefinerte enzymatiske reaksjoner som involverer kollagen hydroksylering, karnitin og noradrenalin biosyntese, tyrosin metabolisme og peptid hormon amidering to. Forbløffende nok montering evidence antyder at askorbat spiller en rolle i å stimulere andre jern-avhengige dioxygenases, slik som prolyl-og asparaginyl hydroksylasene som er involvert i hydroksylering og målretting av den hypoksi-induserbar faktorer (HIFs) 1α og 2α 3.. En fersk rapport antyder at askorbat spiller en rolle i T-celle modning gjennom å påvirke kromatin demetylering via sin aktivitet i stimulere atom hydroksylaser, Jumonji C (JmjC) domene proteiner; sistnevnte som ser ut til å kreve askorbat for full aktivitet 4.. Faktisk, stimulering av slike enzymer ved askorbat synes å forekomme ved en tilsvarende mekanisme for den stimulering av askorbat av HIF-og kollagen-hydroksylasene. Blant andre klassiske virkninger bidrar askorbat vesentlig til cellulær antioxidation som et vannoppløselig kjedebrytende radikal scavenger 5 og til gjenvinning av plasma membran α-tokoferol (vitamin E) via reduksjon av α-tocopheroxyl radikal 6, which er viktig for å beskytte mot membranen lipidperoksidasjon 7.. Viktigere, selv om de fleste pattedyr er i stand til å de novo hepatisk syntese av askorbat fra D-glukose, høyere primater, marsvin og noen flaggermus er avhengig av diettkilder vitamin 8.. Dette er på grunn av inaktivering av Gulo-genet, de orthologues av hvilke i upåvirket pattedyr koder enzymet, γ-gulono-lakton-oksydase 9-13. Dette enzymet er nødvendig for den endelige reaksjon i askorbat biosyntese fra glukose 13. Etter transporter-mediert absorpsjon fra tarm lumen hos mennesker, er askorbat fordelt over hele kroppen av sirkulasjonssystemet. Vitaminet er vanligvis funnet i sin reduserte form ved millimolar konsentrasjoner intracellulært (med unntak av erytrocytter i hvilke konsentrasjoner er typisk lik den rådende plasmakonsentrasjon), og ved mikromolar konsentrations (f.eks 50-200 mm) i de fleste ekstracellulære væsker 14,15. Under fysiologiske forhold, askorbat vanligvis gjennomgår en reversibel ett-elektron oksidasjon til ascorbyl frie radikaler (AFR, også kjent som monodehydroascorbate eller semidehydroascorbate). Mens AFR er et relativt stabilt radikal 16, i fravær av sin raske ett-elektron enzymatisk reduksjon tilbake til askorbat, kan to AFRs ytterligere dismutate til en askorbat og en dehydroascorbate (DHA) 9,13,17. Innenfor det indre av cellen, to-elektron oksydasjonsprodukt av askorbat, DHA, hurtig kan reduseres tilbake til askorbat ved glutation-og NAD (P) H-avhengige enzymatiske og ikke-enzymatiske reaksjoner 13. Mens det er klassisk akseptert at askorbat eneste betydelig rolle i jern metabolisme er å stimulere kosten absorpsjon av ikke-heme jern 18, har vi og andre gitt bevis strongly som tyder på at askorbat spiller en betydelig utvidet rolle i metabolismen av dette metallet. Først, askorbat som er utgitt av askorbat-fylt cellene ser ut til å spille en viktig rolle ved modulering av opptaket av ikke-transferrin-bundet jern av cellene 19,20 og aller siste bevis indikerer at askorbat modulerer også opptaket av transferrin-bundet jern av celler 21, tilsvarer den sistnevnte til en stor fysiologisk jern-opptak rute 22.. Askorbat er viktig for normal sentralnervesystemets funksjon hos pattedyr 23,24. Sammen med binyrebarken, hypofysen, thymus, retina og corpus luteum, inneholder hjernen høye konsentrasjoner av askorbat i forhold til andre kroppsvev 23,25-27. I tillegg er eksponeringen av begge astrocytter 28,29 og neuron-lignende celler 30 til glutamat kjent for å utløse frigjøring av askorbat i det ekstracellulære rom, hvor ascorbate er tenkt å bidra til å beskytte nerveceller mot glutamat-indusert neuronal dysfunksjon 31. Mens den eksakte mekanismen for glutamat-indusert askorbat utgivelse fra astrocytter er ukjent, har vi nylig gitt bevis som indikerer involvering av celle hevelse forårsaket av glutamat opptak av astrocyte glutamat og aspartat transporter (GLAST, også kjent eksitatorisk aminosyre transporter isoformen en [EAAT1 ] hos mennesker), og påfølgende aktivering av volum-sensitive osmolyte-og anion-kanaler (VSOACs) som er gjennomtrengelig for små organiske anioner slik som askorbat 32.. De molekylære identiteter av plasma membran rør involvert i VSOAC formasjon gjenstår å bli identifisert 33,34. Selv om mange analyser har blitt utviklet for bestemmelse av askorbat i biologiske prøver, inkludert spektrofotometriske, fluorometriske og kromatografiske analyser 35,36, er det mye variasjon i spesifisitet, sensitivitet, interference av kjemiske forurensninger, effektiv lineære område og stabilitet av endepunkt analytten. I tillegg andre viktige faktorer som påvirker valg av analysen er hurtighet, brukervennlighet og tilgang til relativt spesialisert utstyr som en høy-ytelse væskekromatografi (HPLC) apparat. Her presenterer vi en enkel og meget spesifikk kolorimetrisk mikro assay for bestemmelse av intracellulær askorbat i dyrkede celler, så vel som en separat analyse for bestemmelse av askorbat-utstrømning fra dyrkede celler. Den sistnevnte analysen tar sikte på å omgå problemet med undervurdering av askorbat utgivelse fra celler på grunn av rask re-opptak av utgitt askorbat av natrium-avhengige askorbat transportører (SVCTs). Selv om begge disse metodene har dukket opp i noen av våre tidligere publikasjoner 19,20,32,37,38, gir dette manuskriptet en eksplisitt instruks og retningslinjer for deres effektiv gjennomføring.

<table border="0" cellpadding="0" cellspacing="0" width="678">
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	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:284px;">Nunc 96-well flat-bottom plates</td>
			<td style="width:135px;">Thermo</td>
			<td style="width:127px;">269620</td>
			<td style="width:132px;">Any flat-bottom 96-well plate can be used</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:284px;">Refrigerated benchtop microcentrifuge</td>
			<td style="width:135px;">Eppendorf&#160;</td>
			<td style="width:127px;">5415D</td>
			<td style="width:132px;">A non-refrigerated microcentrifuge that has been equilibrated to temperature in a cold room can also be used</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:74px;width:284px;">Refrigerated bench-top centrifuge</td>
			<td style="width:135px;">Eppendorf&#160;</td>
			<td style="width:127px;">5810R</td>
			<td style="width:132px;">Swing-bucket</td>
		</tr>
		<tr height="260">
			<td height="260" style="height:260px;width:284px;">Bio-Rad Benchmark Plus Microplate Spectrophotometer</td>
			<td style="width:135px;">Bio-Rad</td>
			<td style="width:127px;"></td>
			<td style="width:132px;">Any microplate spectrophotometer capable of reading at 593 nm can be used and is recommended. If a filter-based plate reader is used, choose the closest wavelength possible and use the standard-curve method.</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:284px;">Ependorf MixMate (microplate orbital mixer)</td>
			<td style="width:135px;">Eppendorf&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:132px;">This is a very versatile and reliable microplate mixer and works very well for these assays</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">General-purpose buffers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Phosphate-buffered saline (PBS), pH 7.4</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:284px;">MOPS-buffered saline (MBS); 137 mM NaCl, 2.7 mM KCl, 15 mM MOPS-Na+, pH 7.3</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">MBS + 5 mM D-glucose (MBS/D)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:284px;">HEPES-buffered saline + 5 mM D-glucose (HBS/D); 137 mM NaCl, 5.2 mM KCl, 1.8 mM CaCl2&#8226;2 H2O, 0.8 mM MgSO4&#8226;7 H2O, 5 mM D-glucose, 20 mM HEPES-Na+, pH 7.3)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Cell permeabilisation buffer (CPB; 0.1% saponin in PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">General chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:284px;">L-ascorbic acid or sodium L-ascorbate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;"></td>
			<td style="width:132px;">Highest purity preparations should be obtained</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:284px;">Dehydro-L-ascorbic acid (DHA) dimer</td>
			<td>Sigma-Aldrich</td>
			<td>30790</td>
			<td style="width:132px;">Aqueous solutions theoretically yield 2 moles of DHA monomer per mole of DHA dimer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:284px;">Cytochalasin B</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">C6762</td>
			<td style="width:132px;">Stock solutions prepared in DMSO or ethanol</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:284px;">Ascorbate oxidase (AO)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">A0157</td>
			<td style="width:132px;">Stock solutions (120 U/ml) can be prepared in PBS or MBS and then frozen in aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Potassium ferricyanide (FIC)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">455989</td>
			<td style="width:132px;">Trihydrate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Ferene-S (3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5&#8242;,5&#8242;&#8242;-disulfonic acid disodium salt)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">92940</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Sodium L-glutamate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">L-glutamine</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Saponin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">47036</td>
			<td style="width:132px;">Prepare a 0.1% stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;"></td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Stock solutions for intracellular ascorbate determination assay</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">3 M sodium acetate (pH 6.0)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Glacial acetic acid</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">0.2 M citric acid</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:284px;">3.3 mM FeCl3 in 0.1 M acetic acid</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">30 mM ferene-S</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">50% (v/v) acetic acid + 30% (w/v) trichloroacetic acid (TCA)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stock solutions for ascorbate-efflux assay</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">AO (120 U/ml)</td>
			<td></td>
			<td></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">2.4 mM ferene-S</td>
			<td></td>
			<td></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">0.12 mM FeCl3 in 0.6 mM sodium-citrate</td>
			<td></td>
			<td></td>
			<td style="width:132px;"></td>
		</tr>
	</tbody>
</table>

a rapid and specific microplate assay for the determination of intra- and extracellular ascorbate in cultured cells

Vitamin C (askorbat) spiller mange viktige roller i cellenes stoffskifte, mange av dem har bare kommet frem i lyset de siste årene. For eksempel, i hjernen, fungerer askorbat i en nervecellene og neuromodulatory måte som innebærer askorbat sykling mellom nevroner og vicinale astrocytter - et forhold som ser ut til å være avgjørende for hjernens askorbat homeostase. I tillegg tyder nye bevis på at askorbat har et sterkt utvidet rolle i å regulere mobilnettet og systemisk jern metabolisme enn er klassisk gjenkjent. Den økende erkjennelse av vesentlig rolle for askorbat i normal og deregulert cellulære og organisme fysiologi krever en rekke mellom gjennomstrømning og høy følsomhet analytiske teknikker som kan utføres uten behov for svært kostbart spesialutstyr. Her gir klare instrukser for en medium-gjennomstrømming, spesifikk og relativt billig mikro assay for bestemmelse av bo vith intra-og ekstracellulært askorbat i cellekultur.

Fastsettelse av intracellulær Askorbat i dyrkede Suspension Cells I den første analyse (fig. 1), er intracellulær askorbat bestemt, følgende askorbat-spesifikk (dvs. AO-sensitive) reduksjon av ferricyanid til ferrocyanid, ved hjelp av meget følsom bestemmelse av ferrocyanid med et tidligere publiserte prosedyre 37.. Påvisning av askorbat er basert på den kolorimetriske chelatering av jern jern som er generert av askorbat-avhengige reduksjon av ferricy...

Watch this Scientific Journal Video about A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells at JoVE.com

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Askorbat spiller mange viktige roller i cellenes stoffskifte, og mange av disse har bare kommet frem i lyset de siste årene. Her beskriver vi en medium-throughput, spesifikk og billig mikroplate-analysen for bestemmelse av både intra-og ekstracellulært askorbat i cellekultur.

En rask og spesifikk Mikro analyse for bestemmelse av Intra-og Ekstracellulær Askorbat i dyrkede celler

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

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