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 taknemmelige for Dr. Stephen Robinson og fru Hania Czerwinska (Monash University) for generøs levering af astrocyt kulturer.

1.. Bestem Intracellulær Ascorbat i dyrkede celler <ol><li> Celledyrkning og høst <ol><li> Grow suspension (f.eks menneskelig erythroleukemia, K562) eller omliggende celler (fx primære astrocytter), ved hjælp af standard kultur procedurer 19-21,32,38. Bemærk: For at sikre celler indeholder ascorbat, indlæse dyrkede celler med ascorbat enten som ascorbat eller DHA 33,39. </li></ol></li><li> Opret en ascorbat indeholdende cellulær ekstrakt ...

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I denne afhandling præsenterer vi to hurtige, konkrete og relativt følsomme kolorimetriske mikroplatte assays til bestemmelse af ascorbat afledt af de intra-og ekstracellulære rum i dyrkede celler. Analyserne kan være afsluttet med adgang til standard laboratorieudstyr og reagenser. Kun moderat dyrt reagens nødvendig for assayet er AO, hvilket er vigtigt, da det giver en høj grad af analyt specificitet mod L-ascorbat. Analyserne er velegnet til enten suspension celler (f.eks...

Opdagelsen af den kemiske natur af ascorbinsyre (vitamin C) og dens identifikation som længe søgte &quot;anti-scorbutic faktor&quot; af Albert Szent-Györgyi og andre i papirer offentliggjort 1928-1934 1 var skelsættende begivenheder i historien i biokemi. Faktisk disse opdagelser har bidraget til Szent-Györgyi blive tildelt Nobelprisen i fysiologi eller medicin i 1937. Den stadig voksende suite af roller for ascorbat i dyre-og plantefysiologi, såvel som menneskers sundhed fortsat være emnerne for aktiv videnskabelig undersøgelse og kontroverser. -L-ascorbat er en rigelig fysiologisk reduktionsmiddel og enzym cofaktor i mammale systemer, og bidrager til en lang række veldefinerede enzymatiske reaktioner, der involverer kollagen hydroxylering, carnitin og noradrenalin biosyntese, tyrosin metabolisme og peptidhormon amidering 2. Interessant montage EVIDEnce foreslår, at ascorbat spiller en rolle i at stimulere andre jern-afhængige dioxygenases, såsom prolyl-og asparaginylrester hydroxylaser involveret i hydroxylering og målretning af hypoxi-inducerbare faktorer (HIFs) 1α og 2α 3. En nylig rapport viser, at ascorbat spiller en rolle i T-celle modning gennem påvirker kromatin demethylering via sin aktivitet i stimulere nukleare hydroxylaser, Jumonji C (JmjC) domæne proteiner; hvor sidstnævnte synes at kræve ascorbat for fuld aktivitet 4. Faktisk, stimulering af sådanne enzymer af ascorbat synes at forekomme ved en lignende mekanisme til stimulering af ascorbat af HIF og collagensygdomme hydroxylaser. Blandt andre klassiske virkninger, ascorbat bidrager væsentligt til cellulære antioxidation som et vandopløseligt kædebrydende radikalrensemiddel 5, og for genvinding af plasmamembranen α-tocopherol (vitamin E) via reduktion af α-tocopheroxyl radikal 6, which er vigtigt at beskytte mod membran lipidperoxidering 7. Vigtigere er det, selv om de fleste pattedyr er i stand til de novo hepatiske syntese af ascorbat fra D-glucose, højere primater, marsvin og nogle flagermus afhænger kostkilder af vitaminet 8. Dette skyldes, at inaktivering af Gulo genet, orthologues hvoraf i upåvirkede pattedyr koder for enzymet, γ-gulono-lacton oxidase 9-13. Dette enzym er nødvendigt for den endelige reaktion i ascorbat biosyntese fra glucose 13. Efter transportør-medieret absorption fra tarmen hos mennesker er ascorbat fordelt i hele kroppen ved kredsløbssygdomme. Vitaminet er typisk findes i sin reducerede form ved millimolære koncentrationer intracellulært (med undtagelse af erythrocytter i hvilke koncentrationer typisk er lig den fremherskende plasma koncentration), og ved mikromolær concentrations (f.eks 50-200 uM) i de fleste ekstracellulære væsker 14,15. Under fysiologiske betingelser ascorbat typisk undergår en reversibel en-elektron-oxidation til ascorbyl frie radikaler (AFR, også kendt som monodehydroascorbate eller semidehydroascorbate). Mens AFR er et relativt stabilt radikal 16, i mangel af sin hurtige one-elektron enzymatisk reduktion tilbage til ascorbat kan to AFRs yderligere dismutate til en ascorbat og én dehydroascorbate (DHA) 9,13,17. Inden i det indre af cellen, to-elektron-oxidation produkt af ascorbat, DHA, kan reduceres hurtigt tilbage til ascorbat ved glutathion-og NAD (P) H-afhængige enzymatiske og ikke-enzymatiske reaktioner 13. Selv om det klassisk er accepteret, at ascorbat eneste væsentlig rolle i jern stofskifte er at stimulere absorptionen af non-hæm jern 18 kosten, har vi og andre fremlagt beviser strongly tyder på, at ascorbat spiller en stærkt udvidet rolle i metabolismen af ​​dette metal. Først ascorbat der er udgivet af askorbatniveauer-propfuld celler synes at spille en vigtig rolle i at modulere optagelsen af non-transferrin-bundet jern af celler 19,20, og meget nyere undersøgelser viser, at ascorbat modulerer også optagelsen af transferrin-bundet jern af celler 21, hvoraf sidstnævnte svarer til en større fysiologisk jern-uptake rute 22. Ascorbat er afgørende for den normale funktion af centralnervesystemet i pattedyr 23,24. Sammen med den adrenale cortex, hypofyse, thymus, retina og corpus luteum, hjernen indeholder høje koncentrationer af ascorbat i forhold til andre kropsvæv 23,25-27. Derudover er eksponering af både astrocytter 28,29 og neuron-lignende celler 30 glutamat kendt for at udløse frigivelse af ascorbat i det ekstracellulære rum, hvor ascorbate menes at hjælpe med at beskytte neuroner mod glutamat-induceret neuronal dysfunktion 31. Mens de nøjagtige mekanisme af glutamat-induceret ascorbate frigivelse fra astrocytter er ukendt, har vi for nylig fremlagt beviser indikerer inddragelse af celle hævelse forårsaget af glutamatoptagelse ved astrocyte glutamat og aspartat transporter (GLAST, også kendt stimulerende aminosyretransporter isoform 1 [EAAT1 ] i mennesker) og deraf følgende aktivering af volumen-følsomme osmolyt og anion-kanaler (VSOACs), der er gennemtrængelig for små organiske anioner såsom ascorbate 32. Den molekylære identitet af plasmamembranen ledningerne involveret i VSOAC dannelse mangler at blive identificeret 33,34. Selv om der er udviklet mange analyser til bestemmelse af ascorbat i biologiske prøver, som omfatter spektrofotometriske, fluorometriske og kromatografiske analyser 35,36, der er meget variation i specificitet, sensitivitet, interference af kemiske forureninger, effektiv lineær rækkevidde og stabilitet endpoint analyt. Derudover andre vigtige faktorer, der påvirker valget af assayet er hurtighed, brugervenlighed og adgang til de relativt specialiseret udstyr, såsom en høj-performance væskekromatografi (HPLC)-apparat. Her præsenterer vi en enkel og meget specifik kolorimetrisk mikroplade-assay til bestemmelse af intracellulære ascorbat i dyrkede celler, såvel som en særskilt analyse til bestemmelse af ascorbat-efflux fra dyrkede celler. Sidstnævnte analyse har til formål at omgå problemet med undervurdering af ascorbat fra celler på grund af hurtig genoptagelse af frigivet askorbat ved natrium-afhængige askorbatniveauer transportører (SVCTs). Selv om begge af disse metoder har optrådt i nogle af vores tidligere udgivelser 19,20,32,37,38, dette manuskript giver en eksplicit sæt af instruktioner og retningslinjer for en effektiv udførelse.

<table border="0" cellpadding="0" cellspacing="0" width="678">
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		<col />
		<col />
		<col />
	</colgroup>
	<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

C-vitamin (ascorbat) spiller mange vigtige roller i cellulære stofskifte, hvoraf mange kun har kommet for dagens lys i de seneste år. For eksempel i hjernen, ascorbat handler på en neuroprotektiv og neuromodulatory måde, der involverer ascorbate cykling mellem neuroner og vicinale astrocytter - et forhold, der synes at være afgørende for hjernens ascorbate homeostase. Derudover nye beviser tyder stærkt på, at ascorbat har en stærkt udvidet rolle i reguleringen af ​​cellulære og systemiske jern stofskifte end er klassisk anerkendt. Den stigende erkendelse af den integrerende rolle i ascorbat i normal og dereguleret cellulære og organismal fysiologi kræver en vifte af medium-throughput og højfølsomme analytiske teknikker, der kan udføres uden behov for meget dyrt specialudstyr. Her giver vi eksplicitte instruktioner til en medium-throughput, specifikke og relativt billig mikroplade assay til bestemmelse af both intra-og ekstracellulære ascorbat i cellekultur.

Bestemmelse af Intracellulær askorbat Dyrkede suspensionsceller I det første assay (figur 1), er intracellulær ascorbat bestemmes efter ascorbat-specifikke (dvs. AO-følsomme) reduktion af ferricyanid til ferrocyanid ved hjælp af den meget følsom bestemmelse af ferrocyanid ved en tidligere offentliggjort procedure 37. Påvisningen af ​​ascorbat er baseret på kolorimetriske chelatering af ferrojern, der genereres af ascorbat-afhængig reduktion af f...

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

En hurtig og konkret Microplate Assay til bestemmelse af intra-og ekstracellulære Ascorbat i dyrkede celler

Ascorbat spiller mange vigtige roller i cellulære stofskifte, hvoraf mange har kun kommet frem i de seneste år. Her beskriver vi en medium-throughput, specifik og billig mikroplade assay til bestemmelse af både intra-og ekstracellulære ascorbat i cellekultur.

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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