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

Siamo grati al dottor Stephen Robinson e la Sig.ra Hania Czerwinska (Monash University) per la generosa offerta di culture astrociti.

1. Determinare ascorbato intracellulare in cellule in coltura <ol><li> Coltura cellulare e la raccolta <ol><li> Grow sospensione (ad esempio eritroleucemia umana, K562) o cellule aderenti (ad esempio astrociti primari) utilizzando le procedure standard di coltura 19-21,32,38. Nota: per garantire le cellule contengono acido ascorbico, caricare cellule in coltura con l&#39;ascorbato sia come ascorbato o DHA 33,39. </li></ol></li><li> Creare un estratto cel...

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In questo articolo presentiamo due test colorimetrici micropiastre rapidi, specifici e relativamente sensibili per la determinazione di ascorbato derivato dai compartimenti intra ed extracellulari nelle cellule in coltura. Le analisi possono essere completate con accesso alla normale attrezzatura da laboratorio e reagenti. Il reagente solo moderatamente costosa necessaria per il dosaggio è AO, che è essenziale in quanto conferisce un elevato grado di specificità analita verso L-ascorbato.</spa...

La scoperta della natura chimica di acido ascorbico (vitamina C), e la sua identificazione come il tanto ricercato &quot;fattore anti-scorbutico&quot;, di Albert Szent-Györgyi e altri in articoli pubblicati 1928-1934 1 sono stati eventi significativi nella storia di biochimica. Infatti, queste scoperte hanno contribuito a Szent-Györgyi stato assegnato il Premio Nobel per la Fisiologia e Medicina nel 1937. La suite in continua espansione dei ruoli per ascorbato di animali e fisiologia vegetale, così come la salute umana, continuano ad essere i soggetti di attivi scientifica indagini e polemiche. L-ascorbato è un abbondante riducente fisiologico e enzima cofattore nei mammiferi, e contribuisce a numerose reazioni enzimatiche ben definiti che coinvolgono il collagene idrossilazione, carnitina e noradrenalina biosintesi, metabolismo della tirosina e ormone peptidico amidazione 2. Curiosamente, Evide di montaggioSNO suggerisce che ascorbato svolge un ruolo nello stimolare altri diossigenasi ferro-dipendenti, come prolina e asparaginyl idrossilasi coinvolti nella idrossilazione e mira dei fattori inducibili (HIF) 1α e 2α 3. Un recente rapporto suggerisce che l&#39;ascorbato svolge un ruolo nella maturazione delle cellule T attraverso colpisce cromatina demetilazione attraverso la sua attività nello stimolare i idrossilasi nucleari, Jumonji C (JmjC) proteine ​​dominio; l&#39;ultimo dei quali sembrano richiedere ascorbato di piena attività 4. Infatti, la stimolazione di tali enzimi da ascorbato sembra verificarsi con un meccanismo simile alla stimolazione da ascorbato delle HIF e collagene idrossilasi. Tra gli altri effetti classici, ascorbato contribuisce significativamente antiossidazione cellulare come una catena-rottura radical scavenger idrosolubile 5 e al riciclo di membrana plasmatica α-tocoferolo (vitamina E) attraverso la riduzione della α-tocopheroxyl radicale 6, which è importante nella protezione contro la perossidazione dei lipidi di membrana 7. È importante sottolineare che, sebbene la maggior parte dei mammiferi sono in grado di sintesi de novo epatica di ascorbato da D-glucosio, primati superiori, cavie e alcuni pipistrelli dipendono da fonti alimentari di vitamina 8. Ciò è dovuto alla inattivazione del gene Gulo, gli ortologhi di cui nei mammiferi affetti codificano l&#39;enzima, γ-gulono-lattone ossidasi 9-13. Questo enzima è necessario per la reazione finale in ascorbato biosintesi di glucosio 13. Dopo l&#39;assorbimento trasportatore mediata dal lume intestinale nell&#39;uomo, ascorbato è distribuito in tutto il corpo attraverso il sistema circolatorio. La vitamina si trova in genere nella sua forma ridotta a millimolari concentrazione intracellulare (con la notevole eccezione di eritrociti in cui le concentrazioni sono in genere simili a concentrazione plasmatica prevalente), e al conc micromolarentrations (ad esempio 50-200 micron) nella maggior parte dei fluidi extracellulari 14,15. In condizioni fisiologiche, ascorbato subisce generalmente reversibile un elettrone ossidazione al ascorbil radicale libero (AFR, noto anche come monodehydroascorbate o semidehydroascorbate). Mentre l&#39;AFR è relativamente stabile radicale 16, in assenza della sua rapida riduzione enzimatica di un elettrone ritorna ascorbato, due AFR può ulteriormente dismutate una ascorbato e una deidroascorbato (DHA) 9,13,17. Entro l&#39;interno della cellula, l&#39;ossidazione del prodotto a due elettroni di ascorbato, DHA, può essere rapidamente ridotta ritorna ascorbato da glutatione-e NAD (P) H enzimatico-dipendente e reazioni non enzimatiche 13. Mentre è classicamente accettato che solo il ruolo significativo di ascorbato nel metabolismo del ferro è di stimolare l&#39;assorbimento alimentare di ferro non-eme 18, noi ed altri abbiamo fornito prove strongly suggerendo che ascorbato gioca un ruolo notevolmente ampliato nel metabolismo di questo metallo. Primo, ascorbato che viene rilasciato da cellule ascorbato-piene sembra giocare un ruolo importante nel modulare l&#39;assorbimento di ferro non legato alla transferrina dalle cellule 19,20, e molto recenti evidenze indicano che l&#39;ascorbato modula anche l&#39;assorbimento di ferro legato alla transferrina da celle 21, l&#39;ultima delle quali corrisponde ad una maggiore fisiologica percorso ferro-uptake 22. L&#39;ascorbato è essenziale per la normale funzione del sistema nervoso centrale dei mammiferi 23,24. Insieme con la corteccia surrenale, ghiandola pituitaria, il timo, retina e corpo luteo, il cervello contiene alte concentrazioni di ascorbato rispetto ad altri tessuti del corpo 23,25-27. Inoltre, l&#39;esposizione di entrambi 28,29 astrociti e cellule simili ai neuroni 30 al glutammato è noto per innescare il rilascio di ascorbato nello spazio extracellulare, dove il ascorbate è pensato per aiutare a proteggere i neuroni contro glutammato indotta da disfunzione neuronale 31. Sebbene l&#39;esatto meccanismo di glutammato indotto ascorbato di liberazione dagli astrociti è sconosciuto, di recente abbiamo fornito prove che indicano il coinvolgimento di cellule gonfiore causato da glutammato assorbimento da parte del glutammato astrociti e aspartato trasportatore (GLAST, noto anche aminoacido eccitatorio trasportatore isoforma 1 [EAAT1 ] nell&#39;uomo) e conseguente attivazione di osmolyte e anionici canali di volume sensibile (VSOACs) che sono permeabili alle piccole anioni organici come ascorbato 32. Le identità molecolari dei condotti membrana plasmatica coinvolti nella formazione VSOAC restano da identificare 33,34. Sebbene molti saggi sono stati sviluppati per la determinazione di ascorbato in campioni biologici, che comprendono spettrofotometriche, cromatografiche fluorimetrica e saggi 35,36, c&#39;è molto variabilità specificità, sensibilità, interference da contaminanti chimici, range lineare efficace e stabilità dell&#39;analita endpoint. Inoltre, altri fattori importanti che influenzano la scelta del saggio sono rapidità, facilità d&#39;uso e l&#39;accesso alle apparecchiature relativamente specializzata come una cromatografia liquida ad alta prestazione (HPLC) apparecchi. Presentiamo qui un saggio colorimetrico per micropiastre semplice e altamente specifico per la determinazione di ascorbato intracellulare in cellule in coltura, così come un test separato per la determinazione di ascorbato-efflusso da cellule coltivate. Il secondo test ha lo scopo di aggirare il problema della sottovalutazione del rilascio ascorbato dalle cellule a causa del rapido re-uptake di ascorbato rilasciato da trasportatori ascorbato di sodio-dipendente (SVCTs). Sebbene entrambi questi metodi sono apparsi in alcune delle nostre precedenti pubblicazioni 19,20,32,37,38, questo manoscritto fornisce un insieme esplicito di istruzioni e linee guida per la loro esecuzione effettiva.

<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

Vitamina C (acido ascorbico) svolge numerosi ruoli importanti nel metabolismo cellulare, molti dei quali sono venuti alla luce solo negli ultimi anni. Ad esempio, all&#39;interno del cervello, ascorbato agisce in maniera neuroprotettivo e neuromodulatori che coinvolge l&#39;ascorbato bicicletta tra i neuroni e astrociti vicinali - un rapporto che sembra essere cruciale per l&#39;ascorbato cervello omeostasi. Inoltre, prove emergenti suggeriscono fortemente che l&#39;ascorbato ha un ruolo notevolmente ampliato nella regolazione del metabolismo del ferro cellulare e sistemica di quanto è riconosciuto in stile classico. La crescente riconoscimento del ruolo fondamentale di ascorbato di fisiologia cellulare e organismal normale e deregolamentato richiede una serie di media-throughput e alta sensibilità tecniche analitiche che possono essere eseguiti senza la necessità di attrezzature specialistiche altamente costosi. Qui forniamo istruzioni esplicite per un mezzo di throughput, specifico e relativamente poco costoso test micropiastra per la determinazione dei both ascorbato intra ed extracellulare nelle cellule in coltura.

Determinazione del ascorbato intracellulare in cellule in sospensione coltivate Nel primo test (Figura 1), ascorbato intracellulare viene determinata, seguendo ascorbato-specifica (cioè AO-sensitive) riduzione del ferricianuro di Ferrocianuro, utilizzando la determinazione altamente sensibile di ferrocianuro da una procedura pubblicata 37. Il rilevamento di ascorbato si basa sulla chelazione colorimetrica del ferro ferroso che viene generato dalla riduzione...

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

Una rapida e specifica Microplate Assay per la determinazione di ascorbato intra-ed extracellulare in cellule in coltura

L&#39;ascorbato interpreta numerosi ruoli importanti nel metabolismo cellulare, molti dei quali sono venuti alla luce solo negli ultimi anni. Qui si descrive un medium-throughput specifico e poco costoso saggio micropiastra per la determinazione di entrambi ascorbato intra ed extracellulare in coltura cellulare.

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

Research

JoVE Journal

Biology

Counting and Determining the Viability of Cultured Cells

Conteggio e determinazione della vitalità delle cellule in coltura

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Ricerca

Biologia

Clonogenic Assay: Adherent Cells

Clonogenica saggio: cellule aderenti

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Isolamento e analisi funzionale dei mitocondri da cellule in coltura e Tissue mouse

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

Rilevazione di intracellulare genica in cellule vive di murini, umana e suina origine utilizzando nanoparticelle di fluorescenza marcata

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

Nefrotossina microiniezione in Zebrafish per creare un modello danno renale acuto

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

L&#39;isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

A Rapid, metodo scalabile per l&#39;isolamento, studio funzionale, e analisi di Cell-derivati ​​matrice extracellulare

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Espressione transitoria e Localizzazione cellulare di proteine ​​ricombinanti in cellule in coltura per insetti

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Analisi di co-immunoprecipitazione utilizzando proteine endogene nucleari da cellule coltivate in condizioni di ipossia

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...