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

We are thankful to Dr Stephen Robinson and Ms Hania Czerwinska (Monash University) for the generous supply of astrocyte cultures.

1. Determine Intracellular Ascorbate in Cultured Cells
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	<li>Cell culture and harvesting
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		<li>Grow suspension (e.g.&#160;human erythroleukemia, K562) or adherent cells (e.g.&#160;primary astrocytes) using standard culture procedures19-21,32,38. Note: to ensure cells contain ascorbate, load cultured cells with ascorbate either as ascorbate or DHA33,39.</li>
	</ol>
	</li>
	<li>Create an ascorbate-containing cellular extract
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		<li>I...

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In this paper we present two rapid, specific and relatively sensitive colorimetric microplate assays for the determination of ascorbate derived from the intra- and extracellular compartments in cultured cells. The assays can be completed with access to standard laboratory equipment and reagents. The only moderately costly reagent required for the assay is AO, which is essential as it imparts a high-degree of analyte specificity toward L-ascorbate. The assays are well suited to either suspen...

The discovery of the chemical nature of ascorbic acid (vitamin C), and its identification as the long-sought &#34;anti-scorbutic factor&#34;, by Albert Szent-Gy&#246;rgyi and others in papers published from 1928 to 19341 were landmark events in the history of biochemistry. Indeed, these discoveries contributed to Szent-Gy&#246;rgyi being awarded the Nobel Prize in Physiology or Medicine in 1937. The ever-expanding suite of roles for ascorbate in animal and plant physiology, as well as human health, continue to be the subjects of active scientific investigation and controversy.
L-Ascorbate is an abundant physiological reductant and enzyme cofactor in mammalian systems, and contributes to numerous well-defined enzymatic reactions involving collagen hydroxylation, carnitine and norepinephrine biosynthesis, tyrosine metabolism and peptide hormone amidation2. Intriguingly, mounting evidence suggests that ascorbate plays a role in stimulating other iron-dependent dioxygenases, such as the prolyl and asparaginyl hydroxylases involved in the hydroxylation and targeting of the hypoxia-inducible factors (HIFs) 1&#945; and 2&#945;3. A recent report suggests that ascorbate plays a role in T-cell maturation through affecting chromatin demethylation via its activity in stimulating the nuclear hydroxylases, Jumonji C (JmjC) domain proteins; the latter of which appear to require ascorbate for full activity4. Indeed, the stimulation of such enzymes by ascorbate appears to occur by a similar mechanism to the stimulation by ascorbate of the HIF and collagen hydroxylases. Among other classical effects, ascorbate contributes significantly to cellular antioxidation as a water-soluble chain-breaking radical scavenger5 and to the recycling of plasma membrane &#945;-tocopherol (vitamin E) via the reduction of the &#945;-tocopheroxyl radical6, which is important in protecting against membrane lipid peroxidation7. Importantly, although most mammals are capable of de novo hepatic synthesis of ascorbate from D-glucose, higher primates, guinea pigs and some bats depend on dietary sources of the vitamin8. This is due to inactivation of the GULO gene, the orthologues of which in unaffected mammals encode the enzyme, &#947;-gulono-lactone oxidase9-13. This enzyme is required for the final reaction in ascorbate biosynthesis from glucose13.
Following transporter-mediated absorption from the intestinal lumen in humans, ascorbate is distributed throughout the body by the circulatory system. The vitamin is typically found in its reduced form at millimolar concentrations intracellularly (with the notable exception of erythrocytes in which concentrations are typically similar to the prevailing plasma concentration), and at micromolar concentrations (e.g.&#160;50-200 &#956;M) in most extracellular fluids14,15 .
Under physiological conditions, ascorbate typically undergoes a reversible one-electron oxidation to the ascorbyl free radical (AFR; also known as monodehydroascorbate or semidehydroascorbate). While the AFR is a relatively stable radical16, in the absence of its rapid one-electron enzymatic reduction back to ascorbate, two AFRs can further dismutate to one ascorbate and one dehydroascorbate (DHA)9,13,17. Within the interior of the cell, the two-electron oxidation product of ascorbate, DHA, can be rapidly reduced back to ascorbate by glutathione- and NAD(P)H-dependent enzymatic and non-enzymatic reactions13.
While it is classically accepted that ascorbate&#8217;s only significant role in iron metabolism is to stimulate dietary absorption of non-heme iron18, we and others have provided evidence strongly suggesting that ascorbate plays a greatly expanded role in the metabolism of this metal. First, ascorbate that is released by ascorbate-replete cells appears to play an important role in modulating the uptake of non-transferrin-bound iron by cells19,20, and very recent evidence indicates that ascorbate also modulates the uptake of transferrin-bound iron by cells21, the latter of which corresponds to a major physiological iron-uptake route22.
Ascorbate is essential for normal central nervous system function in mammals23,24. Together with the adrenal cortex, pituitary gland, thymus, retina and corpus luteum, the brain contains high concentrations of ascorbate relative to other body tissues23,25-27. Additionally, the exposure of both astrocytes28,29 and neuron-like cells30 to glutamate is known to trigger the release of ascorbate into the extracellular space, where the ascorbate is thought to help protect neurons against glutamate-induced neuronal dysfunction31. While the exact mechanism of glutamate-induced ascorbate release from astrocytes is unknown, we have recently provided evidence indicating the involvement of cell swelling caused by glutamate uptake by the astrocyte glutamate and aspartate transporter (GLAST; also known excitatory amino acid transporter isoform 1 [EAAT1] in humans) and consequent activation of volume-sensitive osmolyte and anion channels (VSOACs) that are permeable to small organic anions such as ascorbate32. The molecular identities of the plasma membrane conduits involved in VSOAC formation remain to be identified33,34.
Although many assays have been developed for the determination of ascorbate in biological samples, which include spectrophotometric, fluorometric and chromatographic assays35,36, there is much variability in specificity, sensitivity, interference by chemical contaminants, effective linear range and stability of the endpoint analyte. Additionally, other significant factors that influence the choice of assay are rapidity, ease of use and access to relatively specialized equipment such as a high-performance liquid chromatography (HPLC) apparatus.
Here we present a simple and highly specific colorimetric microplate assay for the determination of intracellular ascorbate in cultured cells, as well as a separate assay for the determination of ascorbate-efflux from cultured cells. The latter assay aims to circumvent the problem of underestimation of ascorbate release from cells due to rapid re-uptake of released ascorbate by sodium-dependent ascorbate transporters (SVCTs). Although both of these methods have appeared in some of our previous publications19,20,32,37,38, this manuscript provides an explicit set of instructions and guidelines for their effective execution.

<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 (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Determination of Intracellular Ascorbate in Cultured Suspension Cells
In the first assay (Figure 1), intracellular ascorbate is determined, following ascorbate-specific (i.e.&#160;AO-sensitive) reduction of ferricyanide to ferrocyanide, using the highly sensitive determination of ferrocyanide by a previously published procedure37. The detection of ascorbate is based on the colorimetric chelation of ferrous iron that is generated by the ascorbate-dependent redu...

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

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

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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