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）洗涤悬浮或贴壁细胞用450微升冰冷的细胞透化缓冲液[CPB适当数量; 0.1％（重量/体积）皂苷的PBS; 4°C]。注意：确定要测定的线性范围内获得吸光度值所需的细胞数量（参见下文）根据经验由最终用户。注：组织提取物也可使用（见讨论进一步的细节）。 </li><li>激越TE细胞于冰上10分钟，以确保彻底的细胞裂解。 </li><li>由粗裂解物离心以16,000×g离心5分钟在冷冻离心（4℃）除去细胞碎片创建一个澄清的含有抗坏血酸胞提取物。 </li><li>小心地从每个样品中取出4×100μl的等分试样，并加入到孔中的一个水平序列中的96孔平底板中，包含任一25微升/孔PBS中只或25微升/孔的45.5 U（“-AO”） / ml的L-抗坏血酸氧化酶（AO）的PBS（“+ AO”）的原液。注意：这应与该标准的制剂并行的进行（见下文）。 </li></ol></li><li> 抗坏血酸标准曲线施工（必须重新构建每个检测） <ol><li>制备10mM的抗坏血酸在冰冷的PBS中的储备液。注：验证抗坏血酸的浓度用分光光度计的石英比色皿用1厘米路径长度在265 nm处（消光系数= 14.5毫米-1厘米-1）40。 </li><li>精心准备了一系列的0和20微米之间的抗坏血酸标准。 </li><li>在步骤1.2.4平行，小心地从每个标准除去4×100μl的等分试样，并添加至孔中，在96孔平底板，其中包含的PBS（“-AO”）或25微升/孔的水平序列45.5 U / ml的储备液AO在PBS（“+ AO”）的。注：100微升的上述抗坏血酸标准将包含0-2纳摩尔抗坏血酸。请注意，该AO的目的是，以控制的非抗坏血酸还原剂以铁氰化钾还原的贡献。 </li></ol></li><li> 确定细胞内抗坏血酸-第1步：选择删除抗坏血酸和定量氧化抗坏血酸与铁氰化钾 <ol><li>在550转通过覆盖板中铜箔轨道式混合96孔板中于室温下放置5分钟，在黑暗中。注：T他的第一步应该氧化抗坏血酸所有在“+ AO”井成DHA。该“-AO”井应该不会受到影响。 </li><li>加入50微升的3.5mM的铁氰化钾（在本文中称为“铁氰化钾”）的PBS至所有孔中。最后的[铁氰化钾] = 1毫米。注：使用multipipettor。 </li><li>在550转轨道式混合的板在室温下搅拌另外5分钟在黑暗中。注意：该步骤应导致铁氰化物的还原由抗坏血酸中的2分子的铁氰化物的每1分子的抗坏血酸的氧化还原的化学计量比，以氰亚铁。 </li><li>立即加入25微升含有50％（V / V）乙酸和30％（重量/体积）三氯乙酸（TCA）新鲜构成的溶液。注：使用multipipettor。 </li></ol></li><li> 确定细胞内抗坏血酸-步骤2：定量的亚铁氰化物的量形成37 <ol><li>加入100微升的亚铁决心解决37至EACH很好。注：工作液应临用前进行：1毫升的3M醋酸钠（pH值6.0）; 0.5毫升冰醋酸（17.4〜2M乙酸）的;将2ml 0.2M的柠檬酸;将2ml 3.3毫米的FeCl 3在0.1M乙酸;将1ml的30mM ferene-S。此工作溶液的最终体积应7.5毫升。 </li><li>在550转轨道式混合板在黑暗中30分钟，在室温下进行。 </li><li>读取各孔的吸光度值在593纳米（ 即最大吸收的铁（II）（ferene-S）3络合物）。 </li><li> &#39; - AO&#39;井对每个样品，然后从抗坏血酸标准曲线内插（见步骤1.3通过最初减去从相应的“+ AO&#39;井在A 593 nm的数值计算细胞内抗坏血酸为每百万个细胞纳摩尔抗坏血酸的量）。对ascorb量每个标准在构建标准曲线，画出这个“差值”每口井吃。 </li></ol></li></ol> 2，从培养细胞中抗坏血酸的测定，外排<ol><li> 细胞培养和收获 <ol><li>长暂停或贴壁细胞上文（见步骤1.1）。注：进行下面的检测与悬挂和贴壁细胞以获得最佳效果24孔平台格式。 </li><li>重悬的细胞悬浮液，或覆盖贴壁细胞，用400μl预热的HEPES缓冲生理盐水溶液，有或没有钙和镁，以及含有5mM D-葡萄糖;在井（HBS / D的pH 7.3，37℃）的24孔板。添加HBS / D相同体积的，在每个24孔板中进行检查指定的无细胞的孔中。注意：后者将作为基线反应无细胞对照组（见下文）。 </li></ol></li></ol> 3，确定维生素C的释放量<ol><li>下面的储备液，应事先准备：120U / ml的AO在HBS / D（准备好新鲜）; 2.4毫米Ferene-S在HBS / D; 120微米的FeCl 3和600μm的柠檬酸钠在HBS / D（立即从比较集中的原液准备）。 </li><li>要启动ferrireduction反应（每孔终体积应为600微升），加试剂已经含有400μLHBS / D的单井以下几卷： <ol><li>加入50微升的AO（120 U / ml）或哈佛商学院/ D的配对井一式三份。最终AO浓度应为10单位/毫升。注意：标号配对孔为“ - AO”和“+ AO”。 </li><li>混合轻轻轨道混合5分钟的板在37°C </li><li>加入50微升2.4毫米ferene-S的所有井。最后ferene-S浓度应为200微米。混合板如上述5分钟，在37℃下</li><li>加入50微升新鲜制备120微米柠檬酸铁的。最终铁和柠檬酸浓度应为10μM铁和50μM的柠檬酸盐。拌匀。 </li></醇&gt; </li><li>三一式三份对照孔，吸上覆介质，并加入600μLHBS /含0.1％皂苷D。注：这些将作为“100％的细胞裂解”控制的乳酸脱氢酶（LDH）释放法，以同步进行（见下文）。 </li><li>孵育在黑暗中60分钟，在37℃下</li><li>在抗坏血酸流出测定法结束时，迅速吸出500μl的各孔，并加入24孔板中，以适当标记的孔中。注意：对于悬浮细胞，首先通过离心在4℃下除去细胞</li><li>加的上清液300μl的等分试样到96孔培养板，然后读取在593纳米。 </li></ol> 4，外测定抗坏血酸<ol><li>读取各孔的吸光度值在593纳米。 </li><li>计算细胞外抗坏血酸的量为每毫克蛋白（或每百万个细胞）纳摩尔抗坏血酸所描述的细胞内抗坏血酸的测定在步骤1.5.4。注意：在最终用户应该优化条件，使抗坏血酸释放不被细胞内抗坏血酸的限制。 </li></ol> 5，测定LDH释放的<ol><li>与剩余的200微升的各样品的细胞外溶液进行LDH释放测定32,41，以确定细胞裂解的程度。注：计算总可释放的LDH的百分比。从分别用0.1％皂素处理的样品确定释放的LDH。 </li></ol>

<ol>
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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume-sensitive+organic+osmolyte/anion+channels+in+cancer:+novel+approaches+to+studying+channel+modulation+employing+proteomics+technologies.">Volume-sensitive organic osmolyte/anion channels in cancer: novel approaches to studying channel modulation employing proteomics technologies.</a> Ann. N.Y. Acad. Sci. 1028, 38-55 (2004).</li><li>Novakova, L., Solich, P., Solichova, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPLC+methods+for+simultaneous+determination+of+ascorbic+and+dehydroascorbic+acids.">HPLC methods for simultaneous determination of ascorbic and dehydroascorbic acids.</a> Trends Anal. Chem. 27, 942-958 (2008).</li><li>Vislisel, J. M., Schafer, F. Q., Buettner, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+sensitive+assay+for+ascorbate+using+a+plate+reader.">A simple and sensitive assay for ascorbate using a plate reader.</a> Anal. Biochem. 365, 31-39 (2007).</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=A+highly+sensitive+colorimetric+microplate+ferrocyanide+assay+applied+to+ascorbate-stimulated+transplasma+membrane+ferricyanide+reduction+and+mitochondrial+succinate+oxidation.">A highly sensitive colorimetric microplate ferrocyanide assay applied to ascorbate-stimulated transplasma membrane ferricyanide reduction and mitochondrial succinate oxidation.</a> Anal. Biochem. 373, 287-295 (2008).</li><li>Lane, D. J. R., Robinson, S. R., Czerwinska, H., 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=A+role+for+Na+/H++exchangers+and+intracellular+pH+in+regulating+vitamin+C-driven+electron+transport+across+the+plasma+membrane.">A role for Na+/H+ exchangers and intracellular pH in regulating vitamin C-driven electron transport across the plasma membrane.</a> Biochem. J. 428, 191-200 (2010).</li><li>Corti, A., Casini, A. F., Pompella, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pathways+for+transport+and+efflux+of+ascorbate+and+dehydroascorbate.">Cellular pathways for transport and efflux of ascorbate and dehydroascorbate.</a> Arch. Biochem. Biophys. 500, 107-115 (2010).</li><li>Laroff, G. P., Fessenden, R. W., Schuler, R. 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>

作者什么都没有透露。

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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			<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>
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			<td></td>
			<td></td>
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			<td height="20" style="height:20px;width:284px;">General-purpose buffers</td>
			<td></td>
			<td></td>
			<td></td>
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		<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>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
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			<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

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。

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

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

a-rapid-specific-microplate-assay-for-determination-intra

Research

JoVE Journal

Biology

13.4K 次观看.  University of Sydney. 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。 

视频: 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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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. ...