Name: a rapid and specific microplate assay for the determination of intra- and extracellular ascorbate in cultured cells
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我们感谢博士斯蒂芬·罗宾逊和女士哈尼亚Czerwinska（莫纳什大学）星形胶质细胞培养的慷慨供应。

1，确定细胞内抗坏血酸在培养细胞<ol><li> 细胞培养和收获 <ol><li>使用标准培养过程19-21,32,38增长悬挂（如人红白血病，K562）或贴壁细胞（ 如原代星形胶质）。注：为确保细胞中含有抗坏血酸，装入培养细胞与抗坏血酸无论是作为抗坏血酸或DHA 33,39。 </li></ol></li><li> 创建一个含有抗坏血酸细胞提取物 <ol><li>孵育磷酸盐缓冲盐水（PBS）洗涤悬...

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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>

在本文中，我们提出了两种快速，特异，灵敏的相对比色微孔板检测抗坏血酸为从培养细胞内和细胞外车厢派生的决心。该测定法可以与访问标准的实验室设备和试剂完成。所需检测的只有适度昂贵的试剂是AO，这是必要的，因为它赋予了高程度的分析物特异性对L-抗坏血酸。的测定是非常适合于任一悬浮细胞（ 如 K562），或贴壁细胞（ 如 HepG2细胞或啮齿动物?...

抗坏血酸（维生素C），而其作为长期追求的“抗坏血病因子”，由阿尔伯特森特- Györgyi和其他人识别的化学性质在公布的1928年至1934年1论文的发现是在历史上具有里程碑意义的事件生物化学。事实上，这些发现有助于森特 - Györgyi被授予诺贝尔生理学或医学，1937年。角色的不断扩展套件，用于抗坏血酸在动物和植物生理学，以及人体健康，继续积极科学的学科调查和争议。 L-抗坏血酸是一种丰富的生理还原剂和在哺乳动物系统中的酶的辅因子，并有助于涉及胶原羟基化，肉碱和去甲肾上腺素的生物合成，酪氨酸代谢和肽激素酰胺2众多良好定义的酶促反应。有趣的是，安装evide考表明抗坏血酸在刺激其他铁依赖性双加氧酶，例如参与羟化脯氨酰和天冬酰胺酰羟化酶和缺氧诱导因子（HIFs）1α和2α3靶向作用。最近的一份报告表明，通过抗坏血酸通过其在刺激核羟化酶，十文字C（JmjC结构）结构域蛋白活性影响染色质甲基化起着T细胞成熟的作用;后者似乎需要对抗坏血酸充分活动4。实际上，这种酶通过抗坏血酸的刺激出现通过类似的机制由HIF和胶原羟化酶抗坏血酸发生的刺激。除其他经典效应，抗坏血酸显著蜂窝抗氧化作为水溶性链破自由基清除剂和5至质膜的再循环有利于α-生育酚通过的α-生育酚自由基06瓦特的减少（维生素E）HICH是防止膜脂过氧化7重要。重要的是，虽然大多数哺乳动物是能够由D-葡萄糖抗坏血酸从头肝脏合成的，高等灵长类动物，豚鼠和一些蝙蝠依赖于维生素8的膳食来源。这是由于在古罗基因失活，其中在未受影响的哺乳动物的同源基因编码的酶，γ-古洛糖酸内酯氧化酶9-13。这种酶是必需的从葡萄糖13中抗坏血酸的生物合成的最终反应。 以下从在人类肠腔转运介导吸收，抗坏血酸是由循环系统分布于整个身体。维生素通常位于其还原形式在毫摩尔浓度细胞内（与红细胞中的浓度通常类似于当时血药浓度的显着的例外），并且在微摩尔浓度entrations（ 如 50-200微米），在大多数细胞外液14,15。 在生理条件下，抗坏血酸盐通常发生可逆单电子氧化的抗坏血酸自由基（AFR，也称为monodehydroascorbate或semidehydroascorbate）。而AFR是一个相对稳定的自由基16，在没有它的快速单电子酶促还原回抗坏血酸，2别生育率可以进一步dismutate到1的抗坏血酸和脱氢抗坏血酸1（DHA）9,13,17。内的细胞内部，抗坏血酸盐，DHA的双电子氧化的产品，可以迅速地通过谷胱甘肽-和NAD（P）H依赖性酶和非酶反应13还原成抗坏血酸。 虽然人们公认的经典，在铁代谢抗坏血酸的唯一显著作用是刺激非血红素铁18饮食吸收，我们和其他人提供的证据strongly表明抗坏血酸在这种金属的代谢中起着很大的作用扩大。首先，抗坏血酸盐，释放由抗坏血酸充满细胞似乎在由细胞19,20调制非转铁蛋白结合的铁的吸收中发挥重要作用，并且非常最近的证据表明，抗坏血酸还调制转铁蛋白-结合的铁的摄取由细胞21，其中后者对应于主要生理铁摄取途径22。 抗坏血酸是用于在哺乳动物23,24正常中枢神经系统的功能是必不可少的。再加上肾上腺皮质，脑垂体，胸腺，视网膜和黄体，大脑中含有高浓度相对于其他身体组织23,25-27抗坏血酸。此外，这两种星形细胞28,29和神经元样细胞30至谷氨酸的曝光是已知的引发抗坏血酸释放到细胞外空间，其中ascorbate的认为有助于保护神经元对谷氨酸诱导的神经元功能障碍31。而从星形胶质细胞谷氨酸诱导的抗坏血酸释放的确切机制尚不清楚，我们最近提供的证据表明细胞肿胀造成摄取谷氨酸的星形胶质细胞谷氨酸和天门冬氨酸转运体（GLAST的参与，也称为兴奋性氨基酸转运蛋白亚型1 [EAAT1在人类中）和体积敏感的渗压剂和阴离子通道（VSOACs）是可渗透的，以小的有机阴离子，如抗坏血酸32后续活化。参与VSOAC形成质膜导管的分子身份有待鉴定33,34。 虽然许多实验已经开发了用于抗坏血酸的生物样品，包括分光光度法，荧光法和层析测定法35,36中的判断，存在特异性，敏感性，干涉的多变性E按化学污染物，有效的线性范围和端点分析物的稳定性。此外，影响测定的选择其它显著因素是快速，易用性和获得相对专业的设备，如高效液相色谱（HPLC）装置。 这里我们提出了细胞内抗坏血酸在培养细胞中的确定，以及对抗坏血酸流出，从培养的细胞中测定的单独测定法的简单和高度特异性的比色微量测定法。后者测定法的目的，以规避从细胞中释放抗坏血酸低估的问题，是由于由钠依赖性抗坏血酸转运体（SVCTs）快速再摄取释放抗坏血酸。虽然这两种方法都出现了一些我们以前的出版物19,20,32,37,38的，该原稿提供了一个明确的指令集，以及用于它们的有效执行的准则。

<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

维生素C（抗坏血酸）在细胞代谢许多重要的角色，其中许多只明朗化的趋势。例如，在大脑中，抗坏血酸的作用在神经保护和神经调节的方式，涉及神经元和星形胶质细胞的邻位之间抗坏血酸循环 - 这似乎是脑抗坏血酸动态平衡至关重要的关系。此外，新的证据有力地表明，抗坏血酸具有调节细胞及全身铁代谢比公认的经典大大扩展作用。人们日益认识到的抗坏血酸在正常和失调的细胞和有机体的生理不可或缺的作用要求的范围内，可无需非常昂贵的专门设备来执行介质通量和高灵敏度的分析技术。在这里，我们提供了一个中等吞吐量明确的指示，为博的决心具体和相对便宜的微孔板检测届内和细胞外抗坏血酸在细胞培养。

在悬浮培养细胞内抗坏血酸的测定在第一试验（ 图1），细胞内抗坏血酸被确定时，以下的抗坏血酸特异性（ 即 AO-敏感）铁氰化钾还原到铁氰化物，使用高灵敏度的测定亚铁氰化物的由先前公布的程序37。抗坏血酸的检测是基于由抗坏血酸依赖性降低铁氰化钾对铁氰化物，随后三价铁的亚铁氰化钾被还原生成二价铁的比色螯合。由于超生理浓度的某?...

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

快速，特异微孔板检测为内和细胞外抗坏血酸在培养细胞的测定

抗坏血酸发挥许多重要作用的细胞代谢，其中许多只明朗化的趋势。这里，我们描述一个介质通量，为在细胞培养物内和细胞外抗坏血酸的确定具体且廉价微孔板检测。

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

计数和确定培养细胞的可行性

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

科研

生物学

Clonogenic Assay: Adherent Cells

克隆形成含量：贴壁细胞

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

隔离和线粒体从培养细胞和小鼠组织功能分析

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

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

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

肾毒素显微注射斑马鱼到模型急性肾损伤

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

核酸的分离过滤：一种简单快速的DNA提取方法

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

一种快速，为隔离，功能可扩展的研究方法，以及细胞衍生的细胞外基质的分析

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

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

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

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

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

缺氧条件下培养细胞内源性核蛋白的共免疫沉淀测定

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