这里报告的研究得到了美国国立卫生研究院国家癌症研究所的部分支持。R33 CA217702和最大化学生发展倡议（IMSD）计划。内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。

人类血清标本是在IRB批准下根据《赫尔辛基宣言》中表达的原则从同意的志愿者那里获得的。在转移到分析实验室之前，对标本进行编码和去鉴定。
1. 样品制备
<ol><li>七氢呋喃二酮细胞裂解物
	<ol>
<li>将每个烧瓶接种约10×10 6 个HepG2细胞放入16个T75烧瓶中，加入14mL EMEM培养基，补充10%胎牛血清（FBS），并培养细胞2天。</li>
		<li>制备 RIPA 缓冲液:在 50 mL 管中，加入 1.5 mL 5 M NaCl、2.5 mL 1 M Tris-HCl （pH = 7）、500 μL NP-40 试剂，然后用 DI 水将最终体积增至 50 mL。</li>
		<li>制备裂解缓冲液:将20mL RIPA缓冲液等分到50mL管中，并加入200μL100x蛋白酶抑制剂溶液以抑制蛋白质和脂质降解。储存在4°C。 注意:裂解缓冲液与TBARS试剂兼容，不会干扰532nm处的吸光度。如果计划使用不同的裂解缓冲液或向裂解缓冲液中添加其他成分，则需要进行初步验证研究以验证裂解缓冲液组分是否与TBARS测定兼容。</li>
		<li>取出含有10%FBS的培养基，用5 mL冷无菌1x PBS洗涤细胞2x。</li>
		<li>向含有细胞的T75烧瓶中加入1mL裂解缓冲液，并在室温（RT）下孵育10分钟，并持续旋转以确保缓冲液分布良好。</li>
		<li>将裂解物收集到适当标记的2 mL卡扣盖聚丙烯管中，并在冰上孵育10分钟。</li>
		<li>在室温下以5，000×g旋转裂解物10分钟以收集细胞碎片，并将上清液吸入单个15 mL管中。</li>
		<li>在50°C和3 mbar下使用Speed Vac将细胞裂解物上清液浓缩四倍，并将94μL的等分试样放入2 mL卡扣帽聚丙烯管中。将样品储存在-80°C，直到用于体外氧化和/或TBARS测定。 注意:为避免浓缩细胞裂解物上清液，也可以使用3mL1x胰蛋白酶分离细胞，用6mL培养基中和，并用5mL冷PBS洗涤2x。然后可以在250μL裂解缓冲液中重构细胞沉淀，然后可以执行步骤1.1.6和1.1.7。</li>
		<li>在乙酸中制备35mM CuCl2 储备溶液（pH = 4）。<ol>
<li>准备乙酸溶液（pH = 4）:将1μL冰醋酸稀释在100mL的DI水中（pH值应约为4，但用pH计确认）。加入更多的水或乙酸，将pH值调节到4。</li>
			<li>取出约0.1936克氯化铜，溶于10毫升乙酸溶液（pH = 4）中，制成144 mM CuCl2 储备液。从该溶液中等分490μL，并加入1，510μL乙酸（pH = 4）以制成35mM CuCl2 溶液。</li>
		</ol>
</li>
		<li>从35 mM CuCl2 储备溶液中等分6μL，并将其加入含有94μL细胞裂解物的6个样品中，使最终CuCl2 浓度约为2mM。将6μL不含CuCl2 的乙酸溶液（pH = 4）加入含有94μL细胞裂解物的六个样品中作为对照。细胞裂解物的最终体积应为100μL，这是用于TBARS测定的体积。 注意:在乙酸（pH = 4）中制造35 mM CuCl2 储备溶液对于防止氢氧化铜沉淀是必要的。</li>
		<li>将样品在37°C的烘箱中孵育24小时，并对含有100μL最终体积的每个样品进行TBARS测定。</li>
		<li>在不同的日子里重复步骤1.1.9和1.1.11 2x，以检查HepG2细胞裂解物的TBARS测定的再现性。</li>
	</ol></li>
	<li>低密度脂蛋白 注意:通常，预纯化的低密度脂蛋白（LDL）含有一定量的EDTA。这里使用的LDL样品含有0.01%的EDTA。EDTA可以抑制体外Cu（II）介导的LDL氧化。因此，在实验或分析之前，可能需要从LDL样品中除去EDTA。步骤 1.2.1–1.2.5 描述了此过程。 注意:氢氧化钠具有腐蚀性，会刺激皮肤和眼睛。使用适当的个人防护装备。<ol>
<li>将24μL从5.51mg / mL低密度脂蛋白储备液（使用BSA作为标准通过改良的Lowry方法测定蛋白质浓度）等分到适当标记的1 mL卡扣帽聚丙烯管中。根据需要制作尽可能多的等分试样，并储存在4°C，直到用于氧化和/或TBARS测定。</li>
		<li>用NaOH微珠在0.15 M NaCl中制备10mM HEPES缓冲液，调节至pH = 7:将4.39gNaCl溶解在0.49L水中，然后加入1.19gHEPES。用搅拌棒充分溶解。加入氢氧化钠珠，直到pH值为7。用水稀释至0.5升。将缓冲液储存在4°C，并在3个月内使用。</li>
		<li>将476μL的10mM HEPES缓冲液加入0.15M NaCl（pH = 7）到等分的LDL样品中，使最终体积达到500μL。将稀释的LDL样品加入具有100K分子量截止值的0.5mL离心离心过滤装置中。</li>
		<li>在室温下以14，000×g离心样品10分钟，留下约30μL的最终截留物体积。将样品在480μL的10mM HEPES缓冲液中在0.15 M NaCl（pH = 7）中重构，并在室温下以14，000×g再次离心10分钟。</li>
		<li>将过滤装置倒置到新的2mL卡扣盖聚丙烯管中，并以1000×g离心2分钟以收集LDL样品（最终体积= 约30μL）。</li>
		<li>将样品等分到适当标记的1 mL管中，并向每个样品中加入20μL水，以达到50μL的最终体积。</li>
		<li>在乙酸中制备200μM CuCl2 储备溶液（pH = 4）<ol>
<li>制备乙酸溶液（pH = 4）:参见步骤1.1.9.1。</li>
			<li>准备144mM CuCl2 储备溶液（见步骤1.1.9.2），然后从144mM CuCl2 储备液中等分5.5μL，溶解在最终体积为4mL乙酸（pH = 4）中以制成200μM溶液。</li>
		</ol>
</li>
		<li>从200μM CuCl2储备溶液中等分2.7μL，并将其加入到含有50μLL的六个样品中，以达到〜10μM的最终CuCl2浓度。将不含CuCl2的乙酸溶液（pH = 4）中的2.7μL加入到含有50μLLL的六个样品中用于对照。</li>
		<li>将LDL样品在37°C的烘箱中孵育2小时。 2小时后，用10mM HEPES缓冲液在0.15M NaCl（pH = 7）中将每个样品的最终体积降至100μL。立即进行 TBARS 检测。</li>
		<li>在两个不同的日期重复步骤1.2.3–1.2.9 2x，以测试TBARS测定的再现性。</li>
	</ol></li>
	<li>人血清
	<ol>
<li>从人血清样品中，将94μL等分试样放入2mL卡扣盖聚丙烯管中，并将样品储存在-80°C。</li>
		<li>在乙酸中制备35mM CuCl2 储备溶液（pH = 4）:参见步骤1.1.9。</li>
		<li>从CuCl2 储备溶液中等分6μL，并将其加入含有94μL人血清的6个样品中，使最终CuCl2 浓度约为2mM。将6μL不含CuCl2 的乙酸溶液（pH = 4）加入含有94μL人血清的六个样品中作为对照使用。</li>
		<li>将人血清样品在37°C的烘箱中孵育24小时，并用TBARS测定法测定氧化水平（第4节）。</li>
		<li>在两天内重复步骤1.3.2–1.3.4 2x，以确定TBARS测定的再现性。</li>
	</ol></li>
</ol>2. 试剂制备
注意:硫代巴比妥酸会引起皮肤和眼睛刺激，吸入或皮肤吸收可能有害。醋酸如果吸入会损害内脏器官。在通风橱中准备所有酸溶液。
<ol><li>制备8.1%（w / v）十二烷基硫酸钠（SDS）溶液<ol>
<li>取出 32.4 g SDS 的重量，溶于烧杯中的 350 mL 去离子水。使用磁性搅拌棒轻轻溶解SDS并避免产生气泡。用去离子水将最终体积调至400 mL，并将SDS溶液储存在室温下。 注意:在这里，准备了过量的8.1%SDS溶液;然而，对于96个样品，只需要约20 mL的8.1%SDS溶液。根据要分析的样品数量准备此溶液。</li>
	</ol>
</li>
	<li>制备3.5M乙酸钠缓冲液（pH = 4）<ol>
<li>在烧杯中将 100 mL 冰醋酸稀释到 350 mL 去离子水中。使用磁性搅拌棒轻轻溶解。</li>
		<li>在水中使用氢氧化钠珠制备6.5M NaOH溶液。将 13 g NaOH 微珠溶解在 40 mL 的 DI 水中，并用 DI 水使最终体积达到 50 mL。</li>
		<li>在与搅拌棒混合时，缓慢地向乙酸溶液中加入约46mL的6.5M NaOH溶液（这应该将pH提高到4，但在使用pH计测量时缓慢加入NaOH溶液来确认）。</li>
		<li>用去离子水将最终体积降至500 mL，并将乙酸钠缓冲液储存在室温下。</li>
	</ol>
</li>
	<li>制备0.8%硫代巴比妥酸水溶液（调节至pH = 4） 注意:在此步骤中，硫代巴比妥酸的制备针对大体积进行了优化，因为将要分析大量样品（108个样品，不包括标准品）。根据计划用于分析的样品数量准备此解决方案。<ol>
<li>使用氢氧化钠珠和水制备5M氢氧化钠溶液:将4g氢氧化钠珠溶于最终体积的20mL水中。存放在塑料容器中。该溶液应为每批新鲜制备。</li>
		<li>重量4克硫代巴比妥酸，加入450毫升去离子水。使用磁性搅拌棒轻轻溶解。 注:该溶液最终将达到500 mL的总体积。</li>
		<li>当用搅拌棒溶解硫代巴比妥酸时，以100μL的增量（缓慢和滴滴方式）加入约3mL的5M NaOH溶液。加入NaOH溶液后，硫代巴比妥酸颗粒将开始溶解。</li>
		<li>如果硫代巴比妥酸颗粒仍未完全溶解，则以100μL的增量加入更多的5M NaOH溶液，直到所有硫代巴比妥酸颗粒完全溶解。对于这种特定体积的溶液，共加入4mL的5M NaOH溶液以完全溶解硫代巴比妥酸颗粒。 注意:在此浓度下，硫代巴比妥酸不会完全溶解，除非pH值接近4。</li>
		<li>在所有硫代巴比妥酸完全溶解后停止添加NaOH。避免超过pH值4。可以通过从混合的硫代巴比妥酸溶液中取1μL并将其置于pH纸上来验证最终的pH值。</li>
		<li>用去离子水将最终体积降至500 mL，并在室温下储存0.8%硫代巴比妥酸水溶液。</li>
	</ol>
</li>
</ol>3. 丙二醛双（缩二甲醇）标准样品制备
注意:丙二醛（MDA）不稳定，无法在市场上买到。然而，市面上有不同的化学形式的MDA，例如MDA四丁基铵盐，MDA双（二甲缩醛）和MDA双（缩二乙醇）。在这三种化学形式中，这里使用MDA双（二甲缩醛），因为大多数研究使用相同的标准21，22。如果选择使用其他两种化学形式的MDA，应事先验证其适用性。
<ol><li>通过将92μL纯MDA双（二甲基缩醛）稀释在1L二甲基苯丙水中，制备550μM MDA双（二甲基缩醛）储备溶液。使用磁性搅拌棒将溶液彻底混合10分钟。将溶液储存在4°C并在1个月内使用。</li>
	<li>通过将550μM MDA双（二甲缩醛）储备液中的726μL稀释到1274μL的DI水中，制备200μM MDA双（二甲缩醛）。每次进行TBARS测定时，应新鲜制备这种200μM MDA双（缩二甲醇）溶液。</li>
	<li>标准曲线制备:取8个2 mL卡扣帽聚丙烯管，并用字母A至H标记，从200μM储备液中加入MDA双（二甲缩醛），并按 表1所述在水中稀释。</li>
	<li>取8个玻璃管（13 mm x 100 mm）并标记它们A-H，然后将100μL标准品加入相应的管中。对空白标准品（样品A）执行六次重复，以计算TBARS测定的检测限。 注意:该协议可以在此处暂停不超过1小时。</li>
</ol>4. TBARS检测
注意:一旦开始TBARS测定，就应该在不停止的情况下完成。
<ol><li>根据需要取尽可能多的玻璃管，以分析样品的数量，并用样品名称标记它们。然后，将100μL每个制备的样品（如上所述）加入每个玻璃管中。</li>
	<li>向每个样品和标准品中加入200μL8.1%SDS，并以圆周运动轻轻旋转玻璃管以混合样品。</li>
	<li>向每个样品和标准品中加入1.5 mL的3.5 M乙酸钠缓冲液（pH = 4）。</li>
	<li>向每个样品和标准品中加入1.5mL的0.8%硫代巴比妥酸水溶液（pH = 4）。</li>
	<li>通过加入700μL的DI水，将每个样品的最终体积和标准品的最终体积降至4 mL。</li>
	<li>紧紧盖住每个玻璃管，并在设置为95°C的加热块中孵育1小时。用铝箔盖住玻璃管，以防止管顶部结露。</li>
	<li>从加热块中取出玻璃管，在冰上孵育30分钟。</li>
	<li>在4°C下以1500×g离心样品和标准品10分钟。 离心后，将含有样品和标准品的玻璃管保持在室温。 注意:将样品保存在冰上或4°C会导致整个样品或标准品沉淀。</li>
	<li>离心后立即，从每个管中等分150μL上清液，并放入96孔板的单独孔中。</li>
	<li>使用移液器吸头从每个孔中除去任何气泡。 注意:气泡的存在会产生不一致的吸光度读数，从而导致高度的测定不精确性。</li>
	<li>读取532nm处的吸光度。从所有其他吸光度读数中减去空白样品的平均吸光度读数。</li>
	<li>通过绘制532nm处的空白减去吸光度读数与每种标准的已知浓度来创建标准曲线。使用线性回归拟合数据点。通过使用从标准曲线获得的线性回归线方程计算未知样品浓度。</li>
</ol>

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作者没有竞争性经济利益或其他利益冲突需要披露。

尽管 TBARS 检测方法存在局限性1、3、4、7、8、9、10、12、13、14、15、19 且不适合在实验室?...

脂质过氧化是自由基（如活性氧和活性氮）攻击脂质中的碳 - 碳双键的过程，该过程涉及从碳中抽取氢并插入氧分子。该过程导致复杂产物的混合物，包括脂质过氧自由基和氢过氧化物作为主要产物，以及丙二醛（MDA）和4-羟基壬烯醛作为主要的二次产物1。
MDA因其与硫代巴比妥酸（TBA）的易反应而被广泛用于生物医学研究中，作为脂质过氧化的标志物。该反应导致MDA-TBA2的形成，MDA-TBA2是一种共轭物，在532nm处的可见光谱中吸收并产生红粉红色2。除MDA外，来自脂质过氧化的其他分子也可以与TBA反应并在532nm处吸收光，从而有助于测量的整体吸收信号。同样，MDA可以与大多数其他主要类别的生物分子反应，可能限制其与TBA3，4反应的可及性。因此，这种传统的测定方法仅被认为可以测量"硫代巴比妥酸反应物质"或TBARS5。
当正确应用和解释时，TBARS测定通常被认为是生物样品中氧化应激总体水平的良好指标6。不幸的是，正如Khoubnasabjafari和其他人所记录的那样，TBARS测定的进行和解释方式往往有助于得出可疑的结论3，4，7，8，9，10，11。造成这种情况的原因主要在于与样品相关的预分析变量，以及缺乏检测坚固性，从而禁止在检测方案中发生看似微小的变化，而不会对测定结果进行实质性更改1，7，12，13。
与生物标本处理和储存相关的预分析变量（例如，暂时保持在-20°C的血浆）14，15 可对TBARS测定结果产生重大影响16，17;如此之多，以至于TBARS测定结果不应在不同实验室之间进行比较，除非有明确的实验室间分析验证数据保证。此建议类似于蛋白质印迹的常用和解释方式。条带密度的比较对于印迹和实验室内研究是有效的，但比较实验室之间的条带密度通常被认为是一种无效的做法。
一些研究人员认为，通过TBARS测定法测量的MDA根本不符合可接受的生物标志物所需的分析或临床标准3，9，10，18，19。事实上，如果该测定法不是在50多年前开发的，它可能不会获得今天的广泛使用和默认接受。尽管还有其他具有更高分析灵敏度、特异性和坚固性的测定方法用于测定氧化应激，但基于532 nm吸光度的 TBARS 测定仍然是迄今为止最常用的测定脂质过氧化的测定方法之一20，从而评估氧化应激。
TBARS测定只能作为昂贵的试剂盒（超过400美元）找到，其中说明书没有提供有关所用试剂的大多数浓度的详细信息。此外，所提供的试剂只能用于一个实验，因为每个试剂盒只能做一个比色标准曲线。对于打算在不同时间点确定几个样品中的氧化水平的研究人员来说，这可能是个问题，因为相同的标准曲线不能多次使用。因此，需要购买多个试剂盒进行多个实验。目前，除非购买昂贵的试剂盒，否则没有有关如何进行TBARS测定的详细方案。过去的一些研究人员模糊地描述了如何进行TBARS测定21，22，但文献中既没有关于如何在没有昂贵试剂盒的情况下进行TBARS测定的完全详细的实验方案或全面的视频。
在这里，我们报告了一种详细的，经过分析验证的用于目的的方法，该方法涉及如何以简单，可重复且廉价的方式进行TBARS测定。用Cu（II）离子处理后人血清，HepG2裂解物和低密度脂蛋白的脂质过氧化变化被证明为TBARS测定的说明性应用。结果表明，这种TBARS测定在日常基础上是一致且可重复的。

<table>
	<tbody>
		<tr>
			<td>1x Sterile PBS pH 7.4 1 L</td>
			<td>VWR, PA</td>
			<td>101642--262</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>50 mL self-standing centrifuge tube</td>
			<td>Corning, NY</td>
			<td>CLS430897</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>96 well plate, Non-Treated, clear, with lid, Non-sterile</td>
			<td>Thermo Fisher Scientific, MA</td>
			<td>280895</td>
			<td>To measure absorbance</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 100 kD centrifugal spin filter device</td>
			<td>Fisher Scientific, NH</td>
			<td>UFC510024</td>
			<td>LDL purification</td>
		</tr>
		<tr>
			<td>Caps for glass tubes</td>
			<td>Thermo Fisher Scientific, MA</td>
			<td>14-930-15D</td>
			<td>for TBARS assay</td>
		</tr>
		<tr>
			<td>Copper II Chloride</td>
			<td>SIGMA, MO</td>
			<td>222011-250G</td>
			<td>to induce oxidation</td>
		</tr>
		<tr>
			<td>Culture tubes, Disposable, with Screw-Cap Finish, Borosilicate Glass (13 x 100 mm)</td>
			<td>VWR, PA</td>
			<td>53283-800</td>
			<td>for TBARS assay</td>
		</tr>
		<tr>
			<td>Eagle&#39;s Minimum Essential Medium (EMEM)</td>
			<td>ATCC, VA</td>
			<td>HB-8065</td>
			<td>HepG2 cell media</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 1.5 mL</td>
			<td>eppendorf, NY</td>
			<td>22363204</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 2.0 mL</td>
			<td>Genesee Sceitific, CA</td>
			<td>22363352</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum US Source</td>
			<td>Omega Scientific, CA</td>
			<td>FB-11</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>SIGMA, MO</td>
			<td>27225-1L-R</td>
			<td>TBARS Reagent</td>
		</tr>
		<tr>
			<td>Halt Protease Inhibitor Cocktail (100x)</td>
			<td>Thermo Scientific, MA</td>
			<td>87786</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SIGMA, MO</td>
			<td>H3375-250G</td>
			<td>LDL solvent</td>
		</tr>
		<tr>
			<td>HepG2 Cells</td>
			<td>ATCC, VA</td>
			<td>HB-8065</td>
			<td>Biological matrix prototype</td>
		</tr>
		<tr>
			<td>Hydrocloric acid (HCl)</td>
			<td>Fisher Scientific, NH</td>
			<td>A144-212</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>Legend Micro 17 Centrifuge</td>
			<td>Thermo Scientific, MA</td>
			<td>75002431</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>Low Density Lipoprotein, Human Plasma</td>
			<td>Athens Research &#38; Technology, GA</td>
			<td>12-16-120412</td>
			<td>Biological matrix prototype</td>
		</tr>
		<tr>
			<td>Magnetic Stir Bars, Octagon 6-Assortment</td>
			<td>VWR, PA</td>
			<td>58948-025</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>Malondialdehyde bis (dimethyl acetal)</td>
			<td>SIGMA, MO</td>
			<td>8207560250</td>
			<td>TBARS Standard</td>
		</tr>
		<tr>
			<td>Multiskan Go Microplate Spectrophotometer</td>
			<td>Fisher Scientific, NH</td>
			<td>51119200</td>
			<td>To measure absorbance</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>EMD Millipore Corp, MA</td>
			<td>492016-100ML</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>SIGMA, MO</td>
			<td>S7653-1KG</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>SIGMA, MO</td>
			<td>436143-100G</td>
			<td>TBARS Reagent</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>SIGMA, MO</td>
			<td>367176-2.5KG</td>
			<td>TBARS Reagent</td>
		</tr>
		<tr>
			<td>SpeedVac Concentrator</td>
			<td>Thermo Scientific, MA</td>
			<td>SC250EXP</td>
			<td>For concentrating cell lysates</td>
		</tr>
		<tr>
			<td>T-75 Flask, Tissue Culture Treated, 250 mL, w/filter cap</td>
			<td>USA Scientific, FL</td>
			<td>658175</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Thiobarbituric Acid</td>
			<td>SIGMA, MO</td>
			<td>T5500-100G</td>
			<td>TBARS Reagent</td>
		</tr>
		<tr>
			<td>TRIS base</td>
			<td>Fluka, GA</td>
			<td>93362</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>Trypsin (1x)</td>
			<td>VWR, PA</td>
			<td>16777-166</td>
			<td>To detach HepG2 cells</td>
		</tr>
	</tbody>
</table>

evaluation of oxidative stress in biological samples using the thiobarbituric acid reactive substances assay

尽管硫代巴比妥酸反应物质（TBARS）测定的分析特异性和坚固性有限，但已被广泛用作生物体液中脂质过氧化的通用指标。它通常被认为是生物样品中氧化应激水平的良好指标，前提是样品已得到妥善处理和储存。该测定涉及脂质过氧化产物（主要是丙二醛（MDA））与硫代巴比妥酸（TBA）的反应，这导致形成称为TBARS的MDA-TBA2加合物。TBARS产生红粉红色，可以在532nm处进行分光光度法测量。TBARS测定在酸性条件（pH = 4）和95°C下进行。 纯MDA是不稳定的，但这些条件允许MDA双（二甲缩醛）释放MDA，MDA在该方法中用作分析标准。TBARS测定是一种简单的方法，可以在大约2小时内完成。此处详细介绍了测定试剂的制备。注重预算的研究人员可以低成本将这些试剂用于多个实验，而不是购买昂贵的TBARS检测试剂盒，该试剂盒仅允许构建单个标准曲线（因此只能用于一个实验）。该TBARS测定的适用性在人血清，低密度脂蛋白和细胞裂解物中显示。该测定结果一致且可重复，检测限可达到 1.1 μM。提供了分光光度法TBARS测定的使用和解释的建议。

在酸性条件（pH = 4）和95°C下，丙二醛（MDA）双（二甲缩醛）产生MDA23。MDA和密切相关的化学同源物与两个硫代巴比妥酸分子（TBA）反应，产生称为硫代巴比妥酸反应物质（TBARS）的化合物，其呈红粉红色，在532nm处具有吸光度λmax （图1， 图2）。以MDA bis（缩二甲醇）为标准，生成标准曲线（图3， ?...

Watch this Scientific Journal Video about Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay at JoVE.com

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

硫代巴比妥酸反应物质测定的目的是通过使用可见光波长分光光度法在532nm处测量脂质过氧化产物（主要是丙二醛）的产生来评估生物样品中的氧化应激。这里描述的方法可应用于人血清、细胞裂解物和低密度脂蛋白。

使用硫代巴比妥酸反应物质测定法评估生物样品中的氧化应激

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic ...

evaluation-oxidative-stress-biological-samples-using-thiobarbituric

Research

JoVE Journal

Chemistry

27.3K 次观看.  Arizona State University. 硫代巴比妥酸反应物质测定的目的是通过使用可见光波长分光光度法在532nm处测量脂质过氧化产物（主要是丙二醛）的产生来评估生物样品中的氧化应激。这里描述的方法可应用于人血清、细胞裂解物和低密度脂蛋白。

视频: Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

科研

化学

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

抗癌金属配合物的合成与细胞毒性评价MTT法

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

Supercritical Nitrogen Processing for the Purification of Reactive Porous Materials

Supercritical fluid extraction and drying methods are well established in numerous applications for the synthesis and processing of porous materials. Herein, nitrogen is presented as a novel supercritical drying fluid for specialized applications such as in the processing of reactive porous materials, where carbon dioxide and other fluids are not appropriate due to their higher chemical reactivity. Nitrogen exhibits similar physical properties in the near-critical region of its phase diagram as compared to carbon dioxide: a widely tunable density up to ~1 g ml-1, modest critical pressure (3.4 MPa), and small molecular diameter of ~3.6 &#197;. The key to achieving a high solvation power of nitrogen is to apply a processing temperature in the range of 80-150 K, where the density of nitrogen is an order of magnitude higher than at similar pressures near ambient temperature. The detailed solvation properties of nitrogen, and especially its selectivity, across a wide range of common target species of extraction still require further investigation. Herein we describe a protocol for the supercritical nitrogen processing of porous magnesium borohydride.

Nitrogen is an effective supercritical fluid for extraction or drying processes due to its small molecular size, high density in the near-liquid supercritical regime, and chemical inertness. We present a supercritical nitrogen drying protocol for the purification treatment of reactive, porous materials.

超临界氮处理的活性多孔材料的净化

Supercritical fluid extraction and drying methods are well established in numerous applications for the synthesis and processing of porous materials. ...

Using Polystyrene-block-poly(acrylic acid)-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are encapsulated in polystyrene-block-poly(acrylic acid) (PSPAA) shells and then used as monomers for their self-assembly. Spherical PSPAA micelles upon acid treatment are known to assemble into cylindrical micelles. Exploiting this tendency, the core-shell nanoparticles are induced to aggregate, coalesce, and then transform into long chains. When more than one type of nanoparticles are used, random and block &#8220;copolymers&#8221; of nanoparticles can be obtained. Detailed procedures are reported for the PSPAA encapsulation of nanoparticles, homo- and co-polymerization of the core-shell nanoparticles, separation and purification of the resulting nanoparticle chains. Transformations of single-line chains into double- and triple-line chains are also presented. The synergy between the polymer shell and the embedded nanoparticles leads to an unusual chain-growth polymerization mode, giving long nanoparticle chains that are distinct from the products of the traditional step-growth aggregation process.

Using Polystyrene-block-polyacrylic acid-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We report protocols for &#8220;polymerizing&#8221; various types of polymer-encapsulated metal nanoparticles into long chains of &#8220;homo-&#8220; and &#8220;co-polymers&#8221;.

使用聚苯乙烯<em&gt;块</em&gt; - 聚（丙烯酸）涂层的金属纳米颗粒作为单体他们均聚物和共聚物聚合

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are ...

Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide (CdSe@CdS) nanorod photocatalysts. Seeded rods are grown using a colloidal hot-injection method and then moved to an aqueous medium by ligand exchange. CdSe@CdS nanorods, an iridium precursor and other salts are mixed and illuminated. The deposition process is initiated by absorption of photons by the semiconductor particle, which results with formation of charge carriers that are used to promote redox reactions. To insure photochemical oxidative growth we used an electron scavenger. The photogenerated holes oxidize the iridium precursor, apparently in a mediated oxidative pathway. This results in the growth of high quality crystalline iridium oxide particles, ranging from 0.5 nm to about 3 nm, along the surface of the rod. Iridium oxide grown on CdSe@CdS heterostructures was studied by a variety of characterization methods, in order to evaluate its characteristics and quality. We explored means for control over particle size, crystallinity, deposition location on the CdS rod, and composition. Illumination time and excitation wavelength were found to be key parameters for such control. The influence of different growth conditions and the characterization of these heterostructures are described alongside a detailed description of their synthesis. Of significance is the fact that the addition of iridium oxide afforded the rods astounding photochemical stability under prolonged illumination in pure water (alleviating the requirement for hole scavengers).

A protocol for the photochemical oxidative growth of small crystalline iridium oxide nanoparticles on the surface of CdSe@CdS seeded rod nanoparticles is presented.

铱氧化物纳米颗粒对硒化镉硫化镉@纳米棒光化学氧化增长

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide ...

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.

A protocol is presented for simultaneously extracting metabolites, proteins, and lipids from a single soil sample, allowing reduced sample preparation times and enabling multi-omic mass spectrometry analyses of samples with limited quantities.

土壤样品多涨价可能分析的 MPLEx 协议

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and ...

Separation of Aldehydes and Reactive Ketones from Mixtures Using a Bisulfite Extraction Protocol

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer by generating a charged structure provides an opportunity to use extraction as a simple purification technique. By combining the use of a miscible organic solvent with saturated sodium bisulfite, aldehydes and reactive ketones can be successfully transformed into charged bisulfite adducts that can then be separated from other organic components of a mixture by the introduction of an immiscible organic layer. Here, we describe a simple protocol for the removal of aldehydes, including sterically-hindered neopentyl aldehydes and some ketones, from chemical mixtures. Ketones can be separated if they are sterically unhindered cyclic or methyl ketones. For aliphatic aldehydes and ketones, dimethylformamide is used as the miscible solvent to improve removal rates. The bisulfite addition reaction can be reversed by basification of the aqueous layer, allowing for the re-isolation of the reactive carbonyl component of a mixture.

Here, we present a protocol to remove aldehydes and reactive ketones from mixtures by a liquid-liquid extraction protocol directly with saturated sodium bisulfite in a miscible solvent. This combined protocol is rapid and facile to perform. The aldehyde or ketone can be re-isolated by the basification of the aqueous layer.

用亚硫酸氢萃取协议分离混合物中的醛和反应性酮

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer ...

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The resulting devices are an example of self-propelled objects capable of generating motion without external actuation and have potential applications in medicine and environmental sciences for a variety of purposes ranging from micro-stirring, targeted therapeutic delivery, to water remediation (e.g., cleaning oil spills). This method employs reactive inkjet printing to generate well-defined small-scale solid silk structures by converting water soluble regenerated silk fibroin (silk I) to insoluble silk fibroin (silk II). These structures are also selectively doped in specific regions with the enzyme catalase in order to produce motion via bubble generation and detachment. The number of layers printed determines the three-dimensional (3D) structure of the device, and so here the effect of this parameter on the propulsive trajectories is reported. The results demonstrate the ability to tune the motion by varying the dimensions of the printed structures.

This protocol demonstrates the ability to utilize reactive inkjet printing to print self-motile biocompatible and environmentally friendly micro-stirrers for use in biomedical and environmental applications.

基于丝绸的自行式微搅拌器的反应式喷墨打印及推进分析

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

硫醇反应试剂的合成与生物共轭作用--用于原位选择性修饰免疫共轭的生成

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.

使用高分辨率X射线吸收光谱在低温下制备生物样品以进行形态分析

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...