我们承认美国国立卫生研究院资助P30ES013508和T32ES019851的支持。

伦理学声明:关于人体标本的处理所有协议遵循美国宾夕法尼亚大学的人类研究伦理委员会的指导方针。 1.缓冲区的制备及100X母液<ol><li>制备1升的碱蒂罗德缓冲液中。结合8.123克氯化钠，1.428克碳酸氢钠，0.466克氯化钙 ∙2 H 2 0，0.224克KCl和0.095克的MgCl 2，调整总体积至1L用的DDH 2 O过滤消毒基地台氏缓冲区。 </li><li>准备葡萄糖和棕榈酸的100倍储备溶液。对于100倍的葡萄糖储备液，溶解100毫克的葡萄糖或[13 C 6]葡萄糖1毫升的DDH 2 O的为100倍，棕榈酸溶液，溶解2.56毫克棕榈酸或在1ml乙醇2.72毫克[13 C 16] -棕榈酸。过滤消毒100X原液。 </li><li>准备适当富集台氏缓冲器<ol&gt; <li>对于非标记研究，加入1毫升的100倍的葡萄糖储备溶液和1ml的100倍的棕榈酸储备溶液至98的基台氏缓冲毫升。 </li><li>葡萄糖标记研究，加入1毫升的100×[13 C 6]葡萄糖储备溶液和1ml的100倍的棕榈酸储备溶液至98的基台氏缓冲毫升。 </li><li>为棕榈酸标记研究，将1ml [13 C 16] -棕榈酸原液的100倍葡萄糖储备溶液和1ml添加到98的基台氏缓冲毫升。 </li></ol></li><li>准备鱼藤酮治疗解决方案<ol><li>制备鱼藤酮在二甲亚砜（DMSO）中的10mM储液。 </li><li>对于非贴标剂量反应的研究，进行1.4.3。对于以单剂量标记研究，继续执行1.4.5。 </li><li>在DMSO执行的10mM鱼藤酮的库存的系列稀释以获得具有鱼藤酮的浓度范围为1nM至100μM的解决方案。这是100倍的股票秒。 </li><li>加入50微升每只股票的5毫升基地台氏缓冲+葡萄糖+棕榈酸从1.3.1准备用鱼藤酮浓度从晚上10点到1微米的解决方案。备选地，添加50微升的DMSO作为载体对照。继续执行2.1。 </li><li>稀释的10mM储备液1,000倍的DMSO以获得10微米的工作溶液。 </li><li>加入50微升这股给每个基地台氏缓冲+5毫升[13 C 6]葡萄糖+棕榈酸从1.3.2和基地台氏缓冲+葡萄糖+ [13 C 16]从1.3.3 -棕榈酸。的最终浓度为100nM的鱼藤酮。另外，加入50微升DMSO的每一个缓冲区，作为车辆的控制。 </li></ol></li></ol> 2.血小板隔离注意:此方法是适合于从全血或血小板从衍生袋血小板。本文中的示例数据准备利用血小板衍生袋血小板。请参见Basu等人 5为使用从全血中分离血小板有关的更多细节。 <ol><li>从全血中分离血小板 注意:如果从血小板一袋血小板分离，继续2.2.1。 <ol><li>离心全血为在没有刹车的15分钟175×g的。 </li><li>传输1毫升上层富血小板血浆（PRP）层到1.5ml微量离心管中。 </li><li>离心在400×g下将PRP 5分钟。 </li><li>吸上清并继续执行步骤3.1或3.2。 </li></ol></li><li>从血小板袋血小板隔离<ol><li>传输1毫升血小板悬浮液至1.5ml微量离心管中。 </li><li>离心在400 xg离心悬浮液5分钟。 </li><li>吸上清并继续执行步骤3.1。或3.2。 </li></ol></li></ol> 3.进行实验<ol><li>为了确定异生的作用治疗（ 例如，鱼藤酮）上感兴趣的代谢物的水平（ 例如，乙酰-CoA），继续3.1.1。来确定到感兴趣的下游代谢产物的代谢前体的掺入生物外源性（ 例如 ，鱼藤酮）的效果，进行3.2.1。 <ol><li>不使用少于三个生物复制为每个条件，从步骤2.1.4或2.2.3在1毫升每种解决方案的步骤1.4.4悬浮颗粒血小板。 </li><li>在水夹套的 CO 2培养箱设定为95％湿度，5％CO 2和37℃下1小时孵育再悬浮血小板。继续执行步骤4.1 注意:这里介绍一个剂量反应实验。或者，时程实验可以做，其中所述剂量是固定和温育的长度是变化的，而不是。 </li></ol></li><li>以确定代谢前体掺入的生物外源性处理的效果到感兴趣的下游代谢产物。 <ol><li> ü唱不超过每个条件的三次生物学重复较少，从步骤2.1.4或2.2.3在标台氏缓冲每个配方1毫升步骤1.4.6悬浮颗粒血小板。 </li><li>在水夹套的 CO 2培养箱设定为95％湿度，5％CO 2和37℃下1小时孵育再悬浮血小板。继续执行步骤4.1。 </li></ol></li></ol> 4.淬火和CoA提取<ol><li>通过以3000 xg离心离心3分钟沉淀血小板。 </li><li>吸上清。 </li><li>在750微升冰冷的10％三氯乙酸悬浮血小板（重量/体积）。 </li><li>添加适当的稳定同位素标记的内部标准。例如，如果目标是辅酶物种的水平跨样品比较，用100微升的稳定同位素如前所述从酵母得到的标辅酶A硫酯（SILEC技术）29的混合物。 </li><li>脉冲声处理每个样品以0.5秒的脉冲30​​次。 </li><li>离心样品以16,000 xg离心在4℃下10分钟。 </li><li>贴上C18固相萃取（SPE）列真空歧管。 </li><li>调节用1ml甲醇的列。 </li><li>平衡柱用1ml的DDH 2 O </li><li>通过列运行样品衍生的上清液（约850微升在这种情况下）。 </li><li>用1毫升清水洗净列。 </li><li>放入10毫升玻璃离心管到真空歧管，收集洗脱部分。 </li><li>用在甲醇中加入1ml的25mM乙酸铵洗脱该柱。 </li><li>在氮气下干燥洗脱液。 </li><li>悬浮干燥残留在50μl5％（重量/体积）的5-磺基水杨酸并转移到HPLC小瓶中。 </li></ol> 5. HPLC安装<ol><li>使用控制软件的HPLC系统中，创建与目标分析物和利用柱进行了优化，如表1中所列的HPLC条件的方法。 注:列TEMPERAT用于该数据的生成URE为25℃，但在特定的参数将通过仪器和相对于感兴趣的化合物而变化。为酰基辅酶A的定量，LC-MS分析的多种方法已被报道，并验证在不同LC条件14,19,20不同的分析物。 </li><li>让HPLC充分平衡。 注意:这应包括溶剂的两个涂刷在该方法的起始溶剂条件附接柱的柱和平衡之前。该平衡容积应按照制造商的指示，以及由此产生的污水应分流到废液。 注:请巴苏和布莱尔5关于HPLC设置的详细信息。 </li></ol> 6.质谱仪设置<ol><li>使用控制软件用于质谱仪，创建用于检测感兴趣的化合物的离子化形式靶向方法。参数介绍在表2中。 注意:这些化合物的稳定同位素类似物应被用来确定一个正或负模式下是否能给予优良的灵敏度和优化源和光学条件为它们的检测。质谱仪方法的总检测时间应该对应于相关的高效液相色谱法的时间分量。 注意:如前面提到的，酰基-CoA分析的多种方法已被报道，包括正面14和负电离15模式和使用高分辨率质谱仪17的，而不是一个三重四极杆仪器28。 </li><li>清洁和校准仪器，建立一个稳定的喷雾进入光谱仪，并且允许时间校准溶液充分从源，并根据制造商的说明光学消散。 </li><li>设置为所有与质谱仪控制软件的样本的序列。 Inclu取消名称或数字标识符，并注明相应的注射量和仪器法。运行的第一个样品之前的空白，并根据需要来减轻残留或基体效应样品之间运行的其他空白和洗涤。 注意:峰积分所选分析物"液体色谱的处理方法，通常可以此顺序内设定或随后在数据处理中应用。 </li><li>开始的顺序和周期性地监视质谱仪和HPLC系统的问题，包括压力波动或系统故障以及在运​​行期间的信号强度的损失。 注:严重问题或持续运行故障可能需要停止运行和吹扫和重新平衡的HPLC系统以及清洁和校准质谱仪。巴苏咨询和布莱尔5关于MS的设置和数据处理的更多细节。 </li></ol>

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Biochem. 474, 59-65 (2015).</li><li>Wallace, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+diseases+in+man+and+mouse.">Mitochondrial diseases in man and mouse.</a> Science. 283 (5407), 1482-1488 (1999).</li><li>Wojtovich, A. P., Brookes, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+II+inhibitor+atpenin+A5+protects+against+cardiac+ischemia-reperfusion+injury+via+activation+of+mitochondrial+KATP+channels.">The complex II inhibitor atpenin A5 protects against cardiac ischemia-reperfusion injury via activation of mitochondrial KATP channels.</a> Basic Res. Cardiol. 104 (2), 121-129 (2009).</li><li>Worth, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotopes+and+LC-MS+for+monitoring+metabolic+disturbances+in+Friedreich's+ataxia+platelets.">Stable isotopes and LC-MS for monitoring metabolic disturbances in Friedreich's ataxia platelets.</a> Bioanalysis. 7 (15), 1843-1855 (2015).</li><li>Worth, A. J., Basu, S. S., Snyder, N. W., Mesaros, C., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+neuronal+cell+mitochondrial+complex+I+with+rotenone+increases+lipid+beta-oxidation,+supporting+acetyl-coenzyme+A+levels.">Inhibition of neuronal cell mitochondrial complex I with rotenone increases lipid beta-oxidation, supporting acetyl-coenzyme A levels.</a> J. Biol. Chem. 289 (39), 26895-26903 (2014).</li><li>Zhou, L., Schmaier, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation+testing+in+platelet-rich+plasma:+description+of+procedures+with+the+aim+to+develop+standards+in+the+field.">Platelet aggregation testing in platelet-rich plasma: description of procedures with the aim to develop standards in the field.</a> Am. J. Clin. Pathol. 123 (2), 172-183 (2005).</li></ol>

作者宣称没有竞争经济利益。

在这里，我们已经表明孤立血小板的效用作为研究扰动线粒体代谢的平台。具体地，我们已响应于鱼藤酮复合物I抑制其特征代谢适应。 目前的研究复杂我抑制细胞系对人类血小板的作用鱼藤酮扩展先前报告的调查结果。重要的是，这已表明，鱼藤酮也抑制血小板琥珀酰-CoA的形成，刺激在血小板βHB辅酶A的增加和对乙酰-CoA的血小板水平没有影响。 极大必须小心，以确保任何此类型的分析的有效性和可重复性。如果血小板将被从全血中分离，如在为了防止淋巴细胞污染和血小板激活上述隔离应完全进行，用专门留下含淋巴细胞undis血沉棕黄层特别注意受扰。为了使剂量响应曲线有意义，连续稀释必须精确地进行，并在每个步骤中的混合至均匀。血小板再悬浮后，孵育条件可以不同于上面详述被修改，但它们必须在整个治疗组中进行严格的标准化，以允许有意义的比较来进行。也许在任何定量的LC / MS分析中最重要的步骤是使用稳定同位素标记的内标。 利用内部标准在此设置非常重要，因为它可以防止潜在的混杂"批量效应"，如可变萃取效率和整个样本分析物的稳定性。内标物加成，（再次，混合至均匀）在实验协议尽早减少工件的可能性。最后，如​​在任何实验中，使用多个复制为每个实验条件是必不可少的，如果可行的话的基础上，中间试验功率计算可能是有用的。 血小板可以从个体或汇集源5,27,32迅速分离，从而使该方法适合于广泛的应用范围。要注意的是，以执行涉及两个相对定量和同位素标记的研究的能力允许对代谢系统的更全面的描述是重要的。目前，这要求两个单独的试验中，这增加了实验所需的生物质的需求。出于这个原因，血小板，其可以大量容易地获得，比其他细胞培养模型更有效。 然而，尽管使用血小板赋予的众多优点，也有缺点。首先，因为有影响血小板生理学25多种疾病必须小心，在人类供体的选择。另外，样品的过程中，血小板活化制备和分析是在任何基于血小板的测定一个潜在的混杂变量。由于这些原因，通过ELISA 26血小板活化的标记物的伴随评估，流式细胞仪1，或测定如光透射集合度（LTA）测定34可以证明谨慎。 虽然本研究的重点是酰基辅酶A硫酯的分析，该方法可以容易地扩展到包括更大范围的分析物。非靶向代谢物组学的方法所提供的机械洞察疾病状态23的阵列。应用这种方法不具有针对性的血小板型号将可能提供更多的洞察涉及失调细胞代谢的基本生化机制。 这种方法可以容易地扩展到包括已知与线粒体代谢干扰其他环境化学品和药物的调查6,12,18,31，以及一个系统，以测试生物异源物质如辅酶A的代谢10的潜在调节剂的效果。另外，一般的方法可用于研究遗传性疾病如弗里德赖希共济失调5,32代谢缺陷。此外，任何上述的实验设计的可以耦合，以便更有力地表征每个上下文2线粒体的状态的功能的呼吸测定。因此，在分离的血小板偶联稳定同位素和LC-MS分析的代谢研究是有可能得到新的机会，以进一步表征异常线粒体代谢。

功能失调线粒体代谢已牵涉多种疾病，包括神经变性，肿瘤和心血管疾病30。这样，巨大的努力已被放在特征有助于疾病的发病机制的代谢缺陷。液相色谱-串联质谱（LC-MS / MS）被认为是用于从复杂的生物基质分析物定量的黄金标准，并经常用于代谢研究8。然而，由于经常与生物医学研究中的情况下，获得有关人类疾病可访问的和明确的模型是一个挑战。 许多研究聘请转化的细胞模型，用于探测生物异源物质或遗传异常的对细胞代谢7,9的影响。发生在肿瘤细胞中可引入混杂代谢重新编程因子21，因此不理想。这些问题可以是circumve原发性细胞模型nted，虽然获得足够的生物质的代谢分析可以是具有挑战性的。此外，大量的文化中使用抗生素的影响已经凸显为潜在的混杂线粒体研究16。 人血小板无力利用与代谢研究5,22,27,32足够的线粒体内容主要细胞模型的机会。首先，血小板可容易地获得的，通过抽血从个体供体，或从血库大量，因此提供其中外部因素可容易地控制的模型。其次，由于它们的尺寸小，血小板可以很容易地从其它血液成分以最小的准备工作中，即使最小装备的实验室5隔离。值得注意的是，血小板不含有晶核，因此可以被用来改变独立地转录调控的研究来代谢。这里，我们表明除了酰基辅酶A（辅酶A）硫酯的相对定量，分离的血小板系统可用于检查碳代谢。具体地讲，我们报道了使用代谢标记的稳定同位素（非放射性）标记的[13 C 6]葡萄糖和[13 C 16] -palmitate探测的[13℃] -label掺入重要代谢产物乙酰经由糖酵解或脂肪酸氧化辅酶A.这提供了一个有力的，概括的，和多功能平台由于酰基-CoA物种的生化途径13,24广泛参与和该系统对测试其他变量，如复合物I的抑制鱼藤酮3,33的易处理性。除了 ​​下面的协议中提供的信息，用于对同位素标记和用于基于LC-MS-分析的方法的广泛的说明可在巴苏和Blair 4中找到。

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Reagent</td>
			<td style="width:92px;"></td>
			<td style="width:68px;"></td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Sodium Chloride (NaCl)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">746398</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Sodium Bicarbonate (NaHCO3)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">S5761</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Calcium Chloride Dihydrate (CaCl2 * H2O)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">223506</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Potassium Chloride (KCl)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">P9541</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">208337</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Glucose</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">G8270</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">13C6-Glucose</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td>389374</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Palmitic acid</td>
			<td style="width:92px;">Cayman</td>
			<td style="width:68px;">10006627</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">13C16-Palmitic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">605573</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Rotenone</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">R8875</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Trichloro Acetic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">T6399</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">5-Sulfosalicylic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">390275</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Acetonitirle</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A996-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Water (H2O)</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">W7-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Formic acid</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">85171</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Dimethyl Sulfoxide</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">472301</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Ethanol</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">04-355-222</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Methanol</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A454-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Ammonium Acetate</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A639-500</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">2 mL Eppendorf Tubes</td>
			<td style="width:92px;">BioExpress</td>
			<td style="width:68px;">C-3229-1</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">LC vials (plastic)</td>
			<td style="width:92px;">Waters</td>
			<td style="width:68px;">186002640</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">10 mL Glass Centrifuge Tubes</td>
			<td style="width:92px;">Kimble Chase</td>
			<td style="width:68px;">73785-10</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Oasis Solid Phase Extraxtion (SPE) Columns</td>
			<td style="width:92px;">Waters</td>
			<td style="width:68px;">WAT094225</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Pastuer Pipets</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">13-678-200</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:161px;">Name</td>
			<td style="width:92px;">Company</td>
			<td style="width:68px;">Catalog Number</td>
			<td style="width:85px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Equipment</td>
			<td style="width:92px;"></td>
			<td style="width:68px;"></td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:161px;">CO2 Water-Jacketed Incubator</td>
			<td style="width:92px;">Nuaire</td>
			<td style="width:68px;">AutoFlow NU-8500</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:161px;">Triple Quadropole Mass Spectrometer</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">Finnigan TSQ Quantum</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:161px;">HPLC</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">Dionex Ultimate 3000</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Source</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">HESI II</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:161px;">HPLC Column</td>
			<td style="width:92px;">Phenomenex</td>
			<td style="width:68px;">Luna C18</td>
			<td style="width:85px;">3 &#956;m particle size, 200 mm x 2 mm</td>
		</tr>
	</tbody>
</table>

lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

为了证明这种方法我们已经再现先前描述代偿代谢适应的所得暴露于鱼藤酮概的效用。这一发现在细胞培养模型先前确定和本调查的目的，可测试此代谢移也发生在血小板，是无核，不容易在相同实验工件作为细胞培养物。这项工作是用6天的血小板宾夕法尼亚创伤中心，已被认为太老了人类输液进行的，虽然他们保留了他们的代谢活动。 如图1中进行了隔离，总的工作流程可以很容易地从临床样品或实验条件的有限数量可以扩展到96孔平板基于更高的吞吐量与血小板不再用于输注到患者有用的可用性。 图2）的抑制导致酰基CoA硫酯的相对水平变化。具体来说，我们在SH-SY5Y细胞观察中琥珀酰-CoA（IC 50 = 25纳米）的剂量依赖性降低，在β羟同步增长（HB）-CoA（ED 50 = 75纳米），而乙酰-CoA水平均持平1。我们与鱼藤酮处理的人血小板的分析表明高度一致的结果（ 图3）。这提供了一个独特的组中指向线粒体的响应于鱼藤酮的依赖人血小板的实验数据。这个响应不能通过常规转录机制，因为血小板缺乏细胞核而介导。 相对定量提供的代谢信息一维的，但同位素标记提供宝贵的互补洞察特定代谢途径的活性。这是在代谢研究非常重要，因为多种途径可通过一个单一的中间会聚。为了证明这一点，血小板用要么为100nM鱼藤酮或DMSO中任一[13 C 6]葡萄糖或[13 C 16] -palmitate的存在下处理1小时。从LC柱酰基辅酶A的同位素的共流出允许最终确定分析物的同位素组成（ 图4）。以下修正为天然丰度重同位素如所述11的这一方法显示在糖酵解生产乙酰-CoA的显著减少血小板而棕榈酸掺入显著增加（ 图5）。这些发现是类似于那些在SH-SY5Y细胞培养模型先前观察到33等提供额外的证据吨帽子此途径可能是体内重要。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>时间（min） </td><td> 0 </td><td> 0 </td><td> 1.5 </td><td>五</td><td> 12 </td><td> 17 </td><td> 18 </td><td> 23 </td><td> 25 </td><td>三十</td></tr><tr><td>总流量（微升/分） </td><td> 200 </td><td> 200 </td><td> 200 </td><td> 200 </td><td> 200 </td><td> 250 </td><td> 200 </td><td> 200 </td><td> 200 </td><td> 200 </td></tr><tr><td> ％ 一个</td><td> 98 </td><td> 98 </td><td> 98 </td><td> 80 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 98 </td><td> 98 </td></tr><tr><td> ％B </td><td> 2 </td><td> 2 </td><td> 2 </td><td> 20 </td><td> 100 </td><td> 100 </td><td> 0 </td><td> 0 </td><td> 2 </td><td> 2 </td></tr><tr><td> ％ C </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td&gt; <td> 0 </td><td> 100 </td><td> 100 </td><td> 0 </td><td> 0 </td></tr></tbody></table> 表 1. 液相色谱梯度溶剂A:5mM的乙酸铵水，溶剂B:5mM的乙酸铵在95:5的ACN /水溶剂C:80:20的ACN /水/0.1%甲酸<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 参数 </td><td> 值 </td></tr><tr><td>喷雾电压（V） </td><td> 4000 </td></tr><tr><td>蒸发器温度（℃） </td><td> 400 </td></tr><tr><td>鞘气压力</td><td> 35 </td></tr><tr><td>辅助气体压力</td><td> 10 </td></tr><tr><td>毛细管温度（℃） </td><td> 350 </td></tr><tr><td>管镜头偏移（V） </td><td> 100 </td></tr></tbody></table><html> 表2.质谱仪参数。 <img alt="图1" src="/files/ftp_upload/53941/53941fig1.jpg" /> 图1.广义工作流样品制备和分析。该示意图描述了由LC-MS / MS分析血小板隔离和治疗代谢研究的工作流程。 <a href="https://www.jove.com/files/ftp_upload/53941/53941fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53941/53941fig2.jpg" /> 图2.葡萄糖代谢方案及脂质代谢和电子传递链。乙酰辅酶A可以从葡萄糖或软脂酸合成。鱼藤酮抑制由线粒体复合物I.电子的正确穿梭TPS://www.jove.com/files/ftp_upload/53941/53941fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53941/53941fig3.jpg" /> 图3.辅酶A硫酯的定量响应于鱼藤酮。鱼藤酮处理不影响乙酰-CoA的水平，但诱导琥珀酰-CoA（IC 50 = 4.1 nm）的与βHB酰-CoA随之增加的剂量依赖性降低（ED 50 = 4.2纳米）。误差棒代表平均值（SEM）的标准误差; N = 4， <a href="https://www.jove.com/files/ftp_upload/53941/53941fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53941/53941fig4.jpg" /> 图4.代表从色谱棕榈显示同位素标记成乙酰辅酶A，鱼藤酮治疗增加了棕榈纳入乙酰CoA。 <a href="https://www.jove.com/files/ftp_upload/53941/53941fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53941/53941fig5.jpg" /> 图5 [13 C 6]葡萄糖或[13 C 16] -palmitate 相对掺入 乙酰-CoA。鱼藤酮处理降低葡萄糖掺入到乙酰-CoA并增加棕榈酸结合。误差棒表示SEM; N = 4，星号表示P &lt;0.05（学生的非配对t检验）。 <a href="https://www.jove.com/files/ftp_upload/53941/53941fig5large.jpg" target="_blank">请点击此处</a>查看该图的放大版本。

Watch this Scientific Journal Video about LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism at JoVE.com

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

这里，我们表明分离的人血小板可以用作一个可访问的体外模型来研究的代谢适应响应于I抑制剂鱼藤酮的复合物。这种方法通过液相色谱 - 质谱法使用同位素示踪和相对定量，并且可以应用于各种研究设计的。

人类血小板的LC-MS分析作为研究线粒体代谢的平台

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

lc-ms-analysis-human-platelets-as-platform-for-studying-mitochondrial

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11.1K 次观看.  University of Pennsylvania. 这里，我们表明分离的人血小板可以用作一个可访问的体外模型来研究的代谢适应响应于I抑制剂鱼藤酮的复合物。这种方法通过液相色谱 - 质谱法使用同位素示踪和相对定量，并且可以应用于各种研究设计的。 

视频: LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

技术树内组织学习节肢动物和微生物群落

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

科研

环境科学

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

使用摇蚊科（双翅目）的表面浮蛹蜕为快速生物议定书水体

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

修改和叶鼓风机-VAC的节肢动物领域采样中的应用

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Thermal Scanning Conductometry (TSC) as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

The thermal scanning conductometry protocol is a new approach in studying ionic gels based on low molecular weight gelators. The method is designed to follow the dynamically changing state of the ionogels, and to deliver more information and details about the subtle change of conductive properties with an increase or decrease in the temperature. Moreover, the method allows the performance of long term (i.e. days, weeks) measurements at a constant temperature to investigate the stability and durability of the system and the aging effects. The main advantage of the TSC method over classical conductometry is the ability to perform measurements during the gelation process, which was impossible with the classical method due to temperature stabilization, which usually takes a long time before the individual measurement. It is a well-known fact that to obtain the physical gel phase, the cooling stage must be fast; moreover, depending on the cooling rate, different microstructures can be achieved. The TSC method can be performed with any cooling/heating rate that can be assured by the external temperature system. In our case, we can achieve linear temperature change rates between 0.1 and approximately 10 &#176;C/min. The thermal scanning conductometry is designed to work in cycles, continuously changing between heating and cooling stages. Such an approach allows study of the reproducibility of the thermally reversible gel-sol phase transition. Moreover, it allows the performance of different experimental protocols on the same sample, which can be refreshed to initial state (if necessary) without removal from the measuring cell. Therefore, the measurements can be performed faster, in a more efficient way, and with much higher reproducibility and accuracy. Additionally, the TSC method can be also used as a tool to manufacture the ionogels with targeted properties, like microstructure, with an instant characterization of conductive properties.

Thermal Scanning Conductometry TSC as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

The kinetics of the cooling process defines the properties of ionic gels based on low molecular weight gelators. This manuscript describes the usage of thermal scanning conductometry (TSC), which obtains full control over the gelation process, along with in situ measurements of the samples' temperature and conductivity.

热扫描电导 (TSC) 作为研究和控制导电物理凝胶相行为的一般方法

The thermal scanning conductometry protocol is a new approach in studying ionic gels based on low molecular weight gelators. The method is designed to ...

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

以玉米 (玉蜀黍) 为例量化植物可溶性蛋白和可消化碳水化合物含量

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

环境污染物对斑马鱼幼虫的神经行为影响研究

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

The Effect of Construction and Demolition Waste Plastic Fractions on Wood-Polymer Composite Properties

Construction and demolition waste (CDW), including valuable materials such as plastics, have a remarkable influence on the waste sector. In order for plastic materials to be re-utilized, they must be identified and separated according to their polymer composition. In this study, the identification of these materials was performed using near-infrared spectroscopy (NIR), which identified material based on their physical-chemical properties. Advantages of the NIR method are a low environmental impact and rapid measurement (within a few seconds) in the spectral range of 1600-2400 nm without special sample preparation. Limitations include its inability to analyze dark materials. The identified polymers were utilized as a component for wood-polymer composite (WPC) that consists of a polymer matrix, low cost fillers, and additives. The components were first compounded with an agglomeration apparatus, followed by production by extrusion. In the agglomeration process, the aim was to compound all materials to produce uniformly distributed and granulated materials as pellets. During the agglomeration process, the polymer (matrix) was melted and fillers and other additives were then mixed into the melted polymer, being ready for the extrusion process. In the extrusion method, heat and shear forces were applied to a material within the barrel of a conical counter-rotating twin-screw type extruder, which reduces the risk of burning the materials and lower shear mixing. The heated and sheared mixture was then conveyed through a die to give the product the desired shape. The above-described protocol proved the potential for re-utilization of CDW materials. Functional properties must be verified according to the standardized tests, such as flexural, tensile, and impact strength tests for the material.

Secondary material streams have been shown to include potential raw materials for production. Presented here is a protocol in which CDW-plastic waste as a raw material is identified, followed by various processing steps (agglomeration, extrusion). As a result, a composite material was produced, and mechanical properties were analyzed.

建筑与拆迁废塑料馏分对木聚合物复合性能的影响

Construction and demolition waste (CDW), including valuable materials such as plastics, have a remarkable influence on the waste sector. In order for ...

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial community assembly. This protocol outlines a system for studying microbial community dynamics in the phyllosphere of Napa cabbage plants. It describes how to grow germ-free plants in test tubes with a calcined clay and nutrient broth substrate. Inoculation of germ-free plants with specific microbial cultures provides opportunities to measure microbial growth and community dynamics in the phyllosphere. Through the use of sterile vegetable extract produced from cabbages shifts in microbial communities that occur during fermentation can also be assessed. This system is relatively simple and inexpensive to set up in the lab and can be used to address key ecological questions in microbial community assembly. It also provides opportunities to understand how phyllosphere community composition can impact the microbial diversity and quality of vegetable fermentations. This approach for developing gnotobiotic cabbage phyllosphere communities could be applied to other wild and agricultural plant species.

A method of growing germ-free Napa cabbages has been developed which enables researchers to evaluate how single microbial species or multispecies microbial communities interact on cabbage leaf surfaces. A sterile vegetable extract is also presented which can be used to measure shifts in community composition during vegetable fermentation.

植物圈和蔬菜发酵微生物群研究的侏儒生物系统

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

微图设计及植物和土壤样品制备，进行15次氮气分析

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...