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
<ol>
	<li>Cell culture and harvesting
	<ol>
		<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
	<ol>
		<li>Incubate an appropriate number of phosphate-buffered saline (PBS)-washed suspension or adherent cells with 450 &#956;l of ice-cold Cell Permeabilization Buffer [CPB; 0.1% (w/v) saponin in PBS; 4 &#176;C]. Note: Determine the number of cells required to obtain an absorbance value within the linear range of the assay (see below) empirically by the end-user. Note: tissue extracts may also be used (see Discussion for further details).</li>
		<li>Agitate cells on ice for 10 min to ensure thorough cellular lysis.</li>
		<li>Create a clarified ascorbate-containing intracellular extract by removing cellular debris by centrifugation of the crude lysate at 16,000 &#215; g for 5 min in a refrigerated microcentrifuge (4 &#176;C).</li>
		<li>Carefully remove 4 x 100 &#956;l aliquots from each sample and add to a horizontal sequence of wells in a 96-well flat bottom plate that contains either 25 &#956;l/well of PBS (&#34;-AO&#34;) only or 25 &#956;l/well of 45.5 U/ml stock solution of L-ascorbate-oxidase (AO) in PBS (&#34;+AO&#34;). Note: this should be done in parallel with the preparation of the standards (see below).</li>
	</ol>
	</li>
	<li>Ascorbate standard-curve construction (must be constructed anew for each assay)
	<ol>
		<li>Prepare a stock solution of 10 mM ascorbic acid in ice-cold PBS. Note: Verify the concentration of ascorbate spectrophotometrically using a quartz cuvette with a 1 cm path-length at 265 nm (extinction coefficient = 14.5 mM-1 cm-1)40.</li>
		<li>Carefully prepare a series of ascorbate standards between 0 and 20 &#956;M.</li>
		<li>In parallel with step 1.2.4, carefully remove 4 &#215; 100 &#956;l aliquots from each standard and add to a horizontal sequence of wells in a 96-well flat bottom plate that contains 25 &#956;l/well of PBS (&#34;-AO&#34;) or a 45.5 U/ml stock solution of AO in PBS (&#34;+AO&#34;). Note: 100 &#956;l of the above ascorbate standards will contain 0-2 nmole of ascorbate. Please note, the purpose of the AO is to control for the contribution of non-ascorbate reductants to ferricyanide reduction.</li>
	</ol>
	</li>
	<li>Determine intracellular ascorbate - Step 1: selectively remove ascorbate and quantitatively oxidize ascorbate with ferricyanide
	<ol>
		<li>Orbitally mix the 96-well plate at 550 rpm at room temperature for 5 min in the dark by covering the plate in foil. Note: This step should oxidize all ascorbate in the &#34;+ AO&#34; wells to DHA. The &#34;-AO&#34; wells should be unaffected.</li>
		<li>Add 50 &#956;l of 3.5 mM potassium ferricyanide (herein referred to as &#34;ferricyanide&#34;) in PBS to all wells. The final [ferricyanide] = 1 mM. Note: Use a multipipettor.</li>
		<li>Orbitally mix the plate at 550 rpm at room temperature for another 5 min in the dark. Note: This step should result in the reduction of ferricyanide to ferrocyanide by ascorbate in a stoichiometric ratio of 2 molecules of ferricyanide reduced per 1 molecule of ascorbate oxidized.</li>
		<li>Immediately add 25 &#956;l of a freshly constructed solution containing 50% (v/v) acetic acid and 30% (w/v) trichloroacetic acid (TCA). Note: Use a multipipettor.</li>
	</ol>
	</li>
	<li>Determine intracellular ascorbate - Step 2: quantitate the amount of ferrocyanide formed37
	<ol>
		<li>Add 100 &#956;l of ferrocyanide determination solution37 to each well. Note: A working solution should be made immediately before use: 2 ml of 3 M Na-acetate (pH 6.0); 0.5 ml of glacial acetic acid (~17.4 M acetic acid); 2 ml of 0.2 M citric acid; 2 ml of 3.3 mM FeCl3 in 0.1 M acetic acid; 1 ml of 30 mM ferene-S. The final volume of this working solution should be 7.5 ml.</li>
		<li>Orbitally mix the plate in the dark at 550 rpm for 30 min at room temperature.</li>
		<li>Read the absorbance values of the wells at 593 nm (i.e.&#160;the absorbance maximum of the Fe(II)(ferene-S)3 complex).</li>
		<li>Calculate the amount of intracellular ascorbate as nmoles ascorbate per million cells by initially subtracting the A593 nm values for the &#39;+ AO&#39; wells from the corresponding &#39;- AO&#39; wells for each sample and then interpolating from the ascorbate standard curve (see step 1.3). When constructing the standard curve, plot this &#34;difference value&#34; for each standard against the amount of ascorbate per well.</li>
	</ol>
	</li>
</ol>
2. Determination of Ascorbate-efflux from Cultured Cells
<ol>
	<li>Cell culture and harvesting
	<ol>
		<li>Grow suspension or adherent cells as above (see step 1.1). Note: carry out the below assay in a 24-well plat format with suspension and adherent cells for best results.</li>
		<li>Resuspend suspension cells, or overlay adherent cells, with 400 &#956;l of pre-warmed HEPES-buffered saline solution, with or without calcium and magnesium, and containing 5 mM D-glucose (HBS/D; pH 7.3, 37 &#176;C) in wells of a 24-well plate. Add the same volume of HBS/D to specified cell-free wells on each 24-well plate to be examined. Note: the latter will serve as cell-free controls for the baseline reaction (see below).</li>
	</ol>
	</li>
</ol>
3. Determine the Amount of Ascorbate Released
<ol>
	<li>The following stock solutions should be prepared beforehand: 120 U/ml AO in HBS/D (prepare fresh); 2.4 mM Ferene-S in HBS/D; 120 &#956;M FeCl3 and 600 &#956;M Na-citrate in HBS/D (prepare immediately from more concentrated stock solutions).</li>
	<li>To start the ferrireduction reaction (final volume per well should be 600 &#956;l), add the following volumes of reagents to individual wells already containing 400 &#956;l of HBS/D:
	<ol>
		<li>Add 50 &#956;l of AO (120 U/ml) or HBS/D to paired wells in triplicate. The final AO concentration should be 10 U/ml. Note: Label paired wells as &#34;- AO&#34; and &#34;+ AO&#34;.</li>
		<li>Mix the plates with gentle orbital mixing for 5 min at 37 &#176;C.</li>
		<li>Add 50 &#956;l of 2.4 mM ferene-S to all wells. The final ferene-S concentration should be 200 &#956;M. Mix the plates as above for 5 min at 37 &#176;C.</li>
		<li>Add 50 &#956;l of freshly prepared 120 &#956;M ferric citrate. The final iron and citrate concentrations should be 10 &#956;M iron and 50 &#956;M citrate. Mix well.</li>
	</ol>
	</li>
	<li>In three triplicate control wells, aspirate the overlying medium and add 600 &#956;l HBS/D containing 0.1% saponin. Note: These will serve as &#34;100% cell-lysis&#34; controls for a lactate dehydrogenase (LDH) release assay to be conducted in parallel (see below).</li>
	<li>Incubate for 60 min in the dark at 37 &#176;C.</li>
	<li>At the end of the ascorbate-efflux assay, rapidly aspirate 500 &#956;l from each well and add to appropriately labelled wells in a 24-well plate. Note: for suspension cells, initially remove the cells by centrifugation at 4 &#176;C.</li>
	<li>Add 300 &#956;l aliquots of the supernatant to a 96-well plate and then read at 593 nm.</li>
</ol>
4. Determination of Extracellular Ascorbate
<ol>
	<li>Read the absorbance values of the wells at 593 nm.</li>
	<li>Calculate the amount of extracellular ascorbate as nmoles ascorbate per mg protein (or per million cells) as described for the determination of intracellular ascorbate in Step 1.5.4. Note: the end user should optimize conditions so that ascorbate release is not limited by intracellular ascorbate.</li>
</ol>
5. Determination of LDH Release
<ol>
	<li>With the remaining 200 &#956;l of extracellular solution from each sample conduct an LDH release assay32,41 to determine the extent of cellular lysis. Note: calculate as a % of total releasable LDH. Determine releasable LDH from the samples that were treated with 0.1% saponin.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electron+spin+resonance+spectra+of+radical+intermediates+in+the+oxidation+of+ascorbic+acid+and+related+substances.">The electron spin resonance spectra of radical intermediates in the oxidation of ascorbic acid and related substances.</a> J. Am. Chem. Soc. 94, 9062-9073 (1972).</li><li>Dringen, R., Kussmaul, L., Hamprecht, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detoxification+of+exogenous+hydrogen+peroxide+and+organic+hydroperoxides+by+cultured+astroglial+cells+assessed+by+microtiter+plate+assay.">Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay.</a> Brain Res. Brain Res. Protoc. 2, 223-228 (1998).</li><li>Lane, D. J. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplasma+membrane+electron+transport+comes+in+two+flavors.">Transplasma membrane electron transport comes in two flavors.</a> Biofactors. 34, 191-200 (2009).</li><li>Lin, S., Lin, D. C., Flanagan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+the+effects+of+cytochalasin+B+on+transport+and+motile+processes.">Specificity of the effects of cytochalasin B on transport and motile processes.</a> Proc. Natl. Acad. Sci. USA. 75, 329-333 (1978).</li><li>May, J. M., Qu, Z. C., Juliao, S., Cobb, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbic+acid+decreases+oxidant+stress+in+endothelial+cells+caused+by+the+nitroxide+tempol.">Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempol.</a> Free Radic. Res. 39, 195-202 (2005).</li><li>Avron, M., Shavit, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+simple+method+for+determination+of+ferrocyanide.">A sensitive and simple method for determination of ferrocyanide.</a> Anal. Biochem. 6, 549-554 (1963).</li></ol>

The authors have nothing to disclose.

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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13.4K Views.  University of Sydney. 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. 

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

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

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

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

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

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

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

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

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

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 decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

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

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

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

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

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

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

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

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

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

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

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