Name: metabolic mapping: quantitative enzyme cytochemistry and histochemistry to determine the activity of dehydrogenases in cells and tissues
Uploaded: 2018-05-26T13:00:00.000+00:00
Duration: 8 min 36 s
Description: Watch this Scientific Journal Video about Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues at JoVE.com

R.J.M. 获得了 AMC 博士奖学金的支持。这项研究得到了荷兰癌症协会 (凯维丰赠款 UVA 2014-6839) 的支持。作者感谢 a. 琼克博士在编写《议定书》方面的帮助。

本议定书遵循学术医学中心医学伦理审查委员会关于使用人体材料的指导方针。
1. 细胞或组织切片的制备
<ol><li>细胞<ol>
<li>胰蛋白酶处理细胞从培养皿使用1毫升0.25% 胰蛋白酶-EDTA 5 分钟在37°c 在10毫米培养皿和带来细胞在37°c 悬浮5000万细胞或 mL, 取决于细胞的大小。</li>
		<li>将细胞悬浮液的200µL 在显微镜下, 使用 cytospin 漏斗在 cytocentrifuge 20 x g 处, 室温5分钟。</li>
		<li>在室温下风干 cytospins 24 小时。这不影响脱氢酶活性。5
</li>
	</ol>
</li>
	<li>组织切片<ol>
<li>根据道德许可, 从病人或动物身上提取组织。10毫米3的组织足以进行多项实验。</li>
		<li>把纸巾放到小通风的2毫升塑料瓶中, 并把含有该片组织的瓶子冷冻在液氮中。</li>
		<li>将含有组织片的小瓶存放在−80°c, 直到进一步加工。</li>
		<li>使用一种称为最佳切削温度 (OCT) 的凝胶介质, 将组织样本装在一个卡盘上, 它可以快速冻结到-20 °c 到-25 摄氏度, 这样水就无法形成晶体。</li>
		<li>使用恒温器切割和首先修剪块到所需的水平 (理想的一半宽度的显微镜玻璃幻灯片, 例如5毫米 x 5 毫米). </li>
		<li>切片时, 使用刷子和防卷板防止部分卷曲向上。</li>
		<li>以缓慢而恒定的速度将组织样品切成6-10 µm 厚的切片 (每节1秒)。当一个截面被正确切割, 它仍然是平的切片刀下的反卷板。</li>
		<li>在显微玻璃幻灯片上捡起1-3 节, 然后在−80°c 储存直到使用。所准备的节数取决于组织样本的大小和所需的截面厚度。</li>
	</ol>
</li>
</ol>2. 酶历史/化学可溶性脱氢酶
<ol><li>准备反应缓冲器。<ol>
<li>准备100毫升磷酸盐缓冲液的正确 ph 值 (请参阅表 1; 每种酶具有最佳的 ph 值, 其活性最高)。例如, 0.1 M 的混合解决方案2PO4 (酸性) 和 0.1 M NA2HPO4 (基本), 直到有正确的 pH 值的缓冲区。</li>
		<li>将聚乙烯醇 (pva) 重18克, 放入烟罩下的磷酸盐缓冲液中, pva 是灰尘和有毒的。<ol>
<li>将18% 瓦特/v PVA 在磷酸盐缓冲器中溶解100摄氏度, 通过升温缓冲au 贝恩玛丽(把玻璃瓶放在沸水中) 搅拌直到 PVA 完全溶解。解决方案将是透明的, 然后, 由搅拌引起的小气泡。它通常需要15分钟的聚乙烯醇溶解。 注: 18% 瓦特/v PVA 在磷酸盐缓冲器是粘性的, 可以转移到15毫升反应缓冲管使用宽吸管。短期贮存18% 瓦特/v PVA 在磷酸盐缓冲应发生在≥40°c 和长期储存 (最多2周) 在≥60摄氏度。250µL 18% 瓦特/v PVA 在磷酸盐缓冲通常需要 cytospin 准备, 而500µL 是需要的一个组织部分。</li>
		</ol>
</li>
		<li>准备四氮唑盐 (nitroBT) 溶液。对于每1毫升的孵化培养基, 需要40µL 的 nitroBT 溶液, 包括5毫克的 nitroBT (黄色粉末) 溶解在20µL 甲基酰胺 (DMF) 和20µL 乙醇, 这将是一个淡黄色透明的解决方案。 注: 由于 nitroBT 的稳定性是所有必要试剂中最低的, 所以建议先准备 nitroBT 的溶液, 然后将 nitroBT 溶解到反应介质中。<ol>
<li>在短时间内 (~ 十年代) 使用本生燃烧器将 nitroBT 在 DMF 和乙醇中溶解。在加热过程中摇动瓶子, 不要在火焰中留下瓶子太长, 以防蒸发。</li>
			<li>做10% 额外的 nitroBT 溶液, 以使 DMF 和乙醇在溶解过程中蒸发。</li>
		</ol>
</li>
		<li>屏蔽电子载体吩 methosulfate (pms) 或 (1-甲氧基)-吩 methosulfate (mPMS), 和孵化媒体含有 m (PMS) 从直接光通过包装锡纸周围的玻璃储存瓶。</li>
		<li>通过在蒸馏 H2O 中溶解它们, 根据表 1准备试剂解决方案。有些试剂很快就会消失, 所以在实验之前尽快准备好。将试剂解决方案保留在冰上或摄氏4摄氏度, 直到使用. </li>
		<li>准备孵化介质, 如表 1中所示, 并在每步经过温和搅拌后融汇解决方案。在搅拌的同时, 要避免在孵化介质中产生气泡, 不断搅拌, 使刮刀保持在孵化培养基的表面下方。</li>
	</ol>
</li>
	<li>孵化培养基在组织切片中的应用<ol>
<li>在免疫组化孵化室中, 用 cytospins 或组织切片附加在37°c 的预温显微镜, 15 分钟. 用温水浸泡在免疫组织化学孵化室的底部, 以保持恒温。孵化器。</li>
		<li>小心地打破或看到了巴斯德吸管在过渡从狭窄到宽的部分。常规的巴斯德吸管太窄, 无法吸入粘稠的孵化培养基, 所以这一步需要使巴斯德吸管足够宽, 以培养培养基的渴望。</li>
		<li>应用250-500 µL 适当的孵化培养基的每一个细胞的准备或组织切片上的显微镜幻灯片使用的广泛部分的巴斯德吸管。</li>
		<li>使用吸管尖端均匀地分散培养培养基。在孵化培养基中忽略小气泡, 因为它们会漂浮到表面, 不会干扰在细胞制备或组织切片上的酶反应。</li>
		<li>根据酶动力学及其在特定细胞制剂中的表达, 在显微镜下的37°c 孵化器 (例如组织培养孵化器) 上孵化细胞制剂或组织切片组织 (表 1)。保持免疫组化孵化室的盖子保持湿润环境, 防止反应缓冲干燥。</li>
	</ol>
</li>
	<li>清洗显微镜幻灯片<ol>
<li>在组织上轻拍多余的孵化培养基。</li>
		<li>机械冲洗的孵化培养基从显微镜幻灯片在60°c 磷酸盐缓冲, pH 值 5.3, 通过移动显微镜在缓冲区上下滑动。这停止了酶的反应。</li>
		<li>通过保持60°c 磷酸盐缓冲液, pH 5.3 为20分钟, 清洗显微镜切片。</li>
		<li>机械清洗显微镜幻灯片在60°c 自来水。</li>
		<li>在60摄氏度自来水中保持20分钟, 以洗涤显微镜的幻灯片。</li>
		<li>让水和幻灯片降温通过慢慢地混合室温自来水与60°c 自来水在1分钟的过程中。</li>
		<li>在室温蒸馏水中执行最后的机械洗涤步骤。</li>
	</ol>
</li>
	<li>将染色的 Cytospins 或组织切片放在显微镜下的幻灯片上<ol>
<li>预热一张幻灯片, 温度在50-60 摄氏度。</li>
		<li>仔细干燥后侧的显微镜幻灯片使用纸巾, 并把他们放在幻灯片上的温暖, 以干的显微镜幻灯片的顶部一侧。</li>
		<li>当幻灯片干燥时, 用甘油果冻安装培养基和盖玻片将细胞制剂或组织切片放在显微镜下的幻灯片上。</li>
		<li>保存细胞制剂或组织切片显微镜幻灯片在黑暗中的冰箱在4摄氏度或立即进行图像采集。</li>
	</ol>
</li>
</ol>3. 图像采集
<ol><li>记录图像与科学的单色 CCD 相机具有足够的动态范围 (至少8位, 但最好10位或12位), 固定增益和线性响应。</li>
	<li>选择一个适当的目标 (20 x-63 x 为 cytospins 和10-63X 的组织切片)。</li>
	<li>通过缩小场膜片来减少眩光, 使其比视野稍大。</li>
	<li>使用单色光, 在 formazan 沉淀7的 isobestic 波长附近应用一个 10 nm 宽带通推理过滤器, 并与红外阻塞过滤器 (请参阅材料表) 进行组合。 注: nitroBT 的最大 isobestic 吸光度为 585 nm。</li>
	<li>优化光照以确保相机的整个灰度范围 (即,设置光照水平, 以便在录制空白显微镜幻灯片时不过度暴露而实现最大光照)。</li>
	<li>校准被测量的灰色水平到已知的吸光度 (或光学密度 [OD]) 值。为此, 捕获至少10个不同步骤的灰度图像, 其中有已知 OD 值的校准幻灯片或步进片 (见步骤 4.1.1), 无论是商用 (参见材料表), 还是测量自定义的灰度楔形步进板, 由densitophotometer 并使用结果将测量的灰色值校准为已知的吸光度值。</li>
	<li>捕获显微照片细胞或组织部分的兴趣。</li>
</ol>4. 图像分析
<ol><li>在图像分析之前, 校准 ImageJ 软件以进行吸光度测量。<ol>
<li>测量具有已知 OD 值的校准滑块或步进片中至少10个校准区域的显微照片的吸光度 (OD) 和已知的吸光度参数。在 ImageJ 中, 使用 "分析→度量值" 菜单或 Ctrl + M 热键测量选定区域。</li>
		<li>通过分析→校准开始吸光度校准程序。选择功能 "Rodbard (NIH 图像)"。在打开的窗口的左列中, 在4.1.1 中执行的校准滑块/步进片的每一步的灰度测量结果已经存在。如果没有, 请从 ImageJ 结果屏幕复制它们。在右列中, 输入与已校准的步骤片所收到的证书上打印的每个相应的已知吸光度值。刻度盒 "全局校准" 和 "显示情节", 并按 "确定"。</li>
		<li>对于质量控制, 请确保 Rodbard 函数的 R2为 > 0.99。如果是 < 0.99, 检查校准幻灯片是否被正确拍照和测量, 以及是否正确输入了已知的吸光度参数。</li>
		<li>ImageJ 现在校准, 直到你关闭程序 (因此, "全球校准")。对于吸光度测量, 校准的 ImageJ 会话总是将观察到的吸光度方法转换为介于0和1之间的值, 其中0和1分别为零和完全吸光度的渐近线。</li>
	</ol>
</li>
	<li>对于 cytospins, 请随机选择 > 100 个单元格。对于组织节, 请在所有节中选择固定长度和宽度的一个或多个感兴趣的字段。<ol>
<li>在显微照片上投射一个光栅, 以帮助不公正的细胞选择。在 ImageJ 中, 通过插件在图像上投影一个网格→分析→网格菜单。</li>
		<li>在 ImageJ 中, 使用椭圆工具选择单个单元格, 并使用矩形工具选择组织区域。</li>
	</ol>
</li>
	<li>测量所选细胞或组织区域的吸光度和面积。在 ImageJ 结果电子表格中, 光密度和面积被称为 "平均值" 和 "面积"。 注意: 细胞或组织区域的总吸光度 (如果你选择了几个不同大小的组织区域) 等于所有分离的像素 absorbances 的总和。如果一个细胞在1000年像素的图像, 那么总吸光度由 1000年 absorbances 的总和和平均吸光度由总 absorbance/1000 给出。此过程还避免了分布错误。</li>
	<li>确定 > 30 细胞或组织区域的平均总吸光度从控制滑块 (s), 其中包含一个样本, 暴露在控制孵化培养基缺乏基质, 但在存在的辅助因子。该控制吸光度用于控制所用显微切片、安装介质、遮盖滑移或显微镜引起的非特异酶活性染色和吸光度制品。</li>
	<li>减去与余子相同浓度 (但不同浓度的基质和/或抑制剂) 接触的样品的所有吸光度测量的平均控制吸收量。这给出了细胞或组织区域的校正总吸光度。</li>
	<li>用学生的 T 检验或单向方差分析测试的平均校正总 absorbances 进行统计学比较, 这取决于你的实验设计。</li>
	<li>Formazan 吸光度可以转化成酶活性 (每毫升µmol 转化的基质) 使用兰伯特-啤酒的定律: c = A/(є×d), 其中 c 是 Formazan 在反应介质中的浓度, A 是校准后 ImageJ 中的测量吸光度;є是 formazan 的消光系数 (16.000 在585毫微米)8和 d 是光的旅行距离, 等于平均细胞厚度或名义切片厚度 (6-10 µm 在这个协议)。 注: 干燥后, 组织断面厚度从公称断面切割厚度减少 50%, 但在上述计算中没有反映, 因为公称断面切割厚度与生物学上最相关。通过共聚焦显微镜或广域显微术, 可以确定 preparates 细胞的平均厚度和球体尺寸, 并可在别处找到协议。9,10
</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enzyme nomenclature 1992 : recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes. , (1992).</li><li>Botman, D., Tigchelaar, W., Van Noorden, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Phosphate-activated+Glutaminase+Activity+and+Its+Kinetics+in+Mouse+Tissues+using+Metabolic+Mapping+(Quantitative+Enzyme+Histochemistry).">Determination of Phosphate-activated Glutaminase Activity and Its Kinetics in Mouse Tissues using Metabolic Mapping (Quantitative Enzyme Histochemistry).</a> J Histochem Cytochem. 62 (11), 813-826 (2014).</li><li>van Lith, S. A., 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=Glutamate+as+chemotactic+fuel+for+diffuse+glioma+cells:+are+they+glutamate+suckers?.">Glutamate as chemotactic fuel for diffuse glioma cells: are they glutamate suckers?.</a> Biochim Biophys Acta. 1846 (1), 66-74 (2014).</li></ol>

作者没有什么可透露的。

对细胞代谢的研究目前正在经历一个新生, 因为研究人员现在意识到新陈代谢对许多疾病的发病机制和治疗是至关重要的。1此外, 新陈代谢研究还得到了越来越多的技术的帮助, 这一领域的研究提供了比以往更多的工具, 包括质谱、放射性同位素标记和核磁共振波谱。新陈代谢图谱绝不是一种新的技术, 但它能够提供综合信息的酶活性在一个几乎真实的情况下, 使这项技术比以往任何时候都更相关。2
在所有活细胞中都发现了辅助因子 NAD + 和 NADP +, 这表明脱氢酶在进化过程中起得很早, 在细胞代谢中起着关键作用. 14,15目前, 有523种酶催化反应被归类为脱氢酶, 并发生在所有物种之间。16理论上, 通过调整此处描述的新陈代谢映射协议, 可以对所有不同脱氢酶反应的活性进行单独的研究。每一种酶反应都是独特的, 通过基质和辅助因子的活动所需的。因此, 每种酶反应的活性都可以通过在反应介质中使用适当基质和辅助因子的代谢图谱实验来确定。然而, 某些酶亚型在其细胞定位上不同,例如一个异构体催化细胞质中的反应, 而另一个异构体在线粒体中起作用。一个显著的例子是 IDH1 和 IDH2, 前者是细胞质, 后者是线粒体, 两种不同的蛋白质编码两个不同的基因。11 1-甲氧基 5-酚嗪 methylsulfate (甲氧基-pms) 和 5-酚嗪 methylsulfate (pms) 可以作为电子载体进行这些实验。前者不通过线粒体膜, 后者。因此, 对线粒体酶 (例如IDH2) 的调查应使用 PMS, 而胞浆酶 (例如IDH1) 的调查应使用 mPMS。
与许多技术一样, 这种协议的变化, 如使用不同类型的四氮唑盐, 不包括增加 PMS 和叠氮化钠, 使用水上安装介质, 和不同的孵化和冲洗时间, 确实存在, 并可能工作同样良好。四氮唑盐代谢图谱的应用不局限于脱氢酶。通过对该协议的小修改, 它也可用于评估代谢通路中脱氢酶直接上游作用的酶活性。我们以前描述了这个原则为 glutaminase 酵素,17直接地作用在谷氨酸脱氢酶的上游在 glutaminolysis 途径。18理论上, 同样的原理也适用于直接作用于脱氢酶例如乌头的其他酶, 它直接位于 tricarboxylic 酸 (TCA) 周期的 IDH2 和 IDH3 上游。表1中所描述的浓度的另一个简单变化是通过使孵化培养基瓶具有不同浓度的基质、余子和/或抑制剂。这允许产生剂量-活动曲线作为一个功能的基质, 余子和/或抑制浓度。
从技术上讲, 新陈代谢图谱实验不利于对原位酶活性的无偏观测, 因为研究人员必须选择一种基质和余子浓度, 而这些物质可能不反映基质和余子水平。就地显示。此外, 在反应介质中使用粘性 18% PVA 有助于使大分子保持完好、到位和构象, 但禁止通过反应介质有效地扩散低分子量试剂。3,5因此, 在这里描述的实验中, 使用 supraphysiological 基质浓度的确定酶活性并不能反映给定基底浓度的体内情况, 但适合于实验内比较。因此, 代谢图谱实验的结果是在所述基质和余子水平的上下文中的酶反应的生产能力 (最大活性), 目前是原位。此外, 使用不同浓度的基质和/或辅助因子和多个样本允许无偏比较的最大生产能力 (V max) 和亲和 (K m) 的酶为基板/余子的细胞制剂、组织或组织区域。这些参数是酶蛋白表达的总和, 转化后的修饰和拥挤的微环境对酶活性的影响。因此, 代谢图谱仍然是一个更好的反映酶活性比实验, 确定蛋白表达或纯化酶活性的在体外。

细胞生理学的基本基石是新陈代谢。新陈代谢为细胞提供了所有生理过程所需的能量, 提供了大分子生物合成的基石, 并对废物、有毒分子和拆卸的细胞稳态进行了调节, 并循环利用不必要或功能失调的细胞成分。酶催化几乎所有重要的细胞化学反应, 因此是细胞生理学的运动单位。1,2
酶活性在许多层次上严格调节, 因此定量酶组织化学和化学 (也称为代谢图谱) 是研究酶活性的首选方法原位。在转录水平, 基因表达到 mRNA 被调控。转录调节的作用可以通过定量 mrna 测定 (如微阵列或直接 RNA 测序) 或定性 mRNA 检测 (如原位杂交) 来确定, 提供有关 (子) 细胞的信息。mRNA 分子的定位。这使人们有可能了解细胞间转录活动的相对差异。然而, 这些 mrna 检测对代谢活动的解释和有效性是复杂的, 因为调控也发生在 mrna 水平, 其中 mrna 分子的序列和稳定性编辑后, 转录从 DNA。这个编辑调控蛋白质异构体翻译和蛋白质翻译数量在核糖体。此外, 蛋白质的翻译过程被控制, 最终影响酶的表达, 因此活动。使用定量蛋白质表达方法, 如西方印迹或反相蛋白裂解微阵列, 或定性蛋白表达法, 可以欣赏上述调控步骤的综合效果, 如免疫细胞化学和免疫组化, 但这些技术并没有纳入下游的调控作用, 如后转化的蛋白质和功能调节的酶在其拥挤的微环境。此外, 酶的表达可能与其活性差, 因此, 在细胞或组织组织匀浆或稀释溶液中的纯化酶活性测量被广泛用于酶活性研究。然而, 这些实验未能复制一个拥挤的分裂细胞质或细胞器对酶活性的影响。此外, 上述所有技术都决定了 mRNA 或酶表达的数量或本地化, 但无法提供关于酶表达的这两个方面的综合信息, 更不用说这种信息以酵素活动决心。2,3,4
代谢图谱允许对所有上述变量的评价, 以确定酶的活性。更重要的是, 代谢图谱是一种活细胞或组织成像的细胞和组织在分析期间保持完好, 以产生一个几乎真实的情况, 以产生酶活性的数据。它产生的图像, 既有利于对酶活性的位置的详细评价, 以及在细胞或组织室内的酶活性的稳健量化。3,4此处描述的脱氢酶活动代谢映射的协议基于所有可用酶组织化学方法的实验室手册, 5 关于定量酶组织化学的原理, 6 和 定量代谢图谱的图像分析。4
脱氢酶是酶, 执行氧化还原反应, 以减少标准辅助因子, 如NAD+,NADP + 和时尚 NADH, 和 FADH 2 分别.未化学固定的完整细胞或恒温器组织切片, 在反应介质中孵化, 其中含有特定脱氢酶和四氮唑盐的基质和辅助因子。随后, 该脱氢酶执行其催化活性, 并减少, 例如, NADP+ 。通过电子载体, 2 分子最终将1水溶性半无色四氮唑盐分子减少为1水溶性蓝 formazan 分子, 立即沉淀在脱氢酶的部位。因此, 沉淀 formazan 的吸光度是一种直接测量脱氢酶局部活性的方法, 可以用光镜观察或用单色显微镜和图像分析的方法进行量化。这个协议的数量方面使研究人员能够从这些化验中得出统计结论。此外, 这种量化有助于确定原位的动力学酶参数, 如最大酶活性 (V最大) 和亲和性的基质为酶 (Km)。3,4,5,6
在规划一个代谢图谱实验时, 重要的是要认识到, 每一个实验, 建议最多五十玻片与黏附细胞制剂或组织切片, 以尽量减少时间延迟孵化期间, 以获得一致的结果。当这些协议步骤由多个实验者协同执行时, 可以处理更多的幻灯片和/或介质组合。此外, 实验之间存在一些变异性。因此, 相同的实验应重复至少3次与 cytospins 从每个细胞的准备或切片从每个组织样本和适当的控制应该包括在每个实验。在没有基质的情况下, 总是准备一个控制孵化培养基, 但在辅助因子的存在下, 控制非特异酶活性染色。最有代表性的结果是在实验中获得了不同的基质/余子浓度被应用于组织切片或细胞制剂从同一样本, 使实验者可以进行酶动力学分析, 利用剂量-活动曲线。

<table><tbody><tr><td>Adenosine 5&amp;prime;-diphosphate sodium salt</td><td>Sigma-Aldrich</td><td>A2754</td><td></td></tr><tr><td>D-Glucose 6-phosphate sodium salt</td><td>Sigma-Aldrich</td><td>G7879</td><td></td></tr><tr><td>Disodium hydrogen phosphate (NA&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Merck Millipore</td><td>106559</td><td></td></tr><tr><td>di-Sodium succinate</td><td>Sigma-Aldrich</td><td>W327700</td><td></td></tr><tr><td>DL-Isocitric acid trisodium salt hydrate</td><td>Sigma-Aldrich</td><td>I1252&amp;nbsp;</td><td></td></tr><tr><td>D-Malic acid</td><td>Sigma-Aldrich</td><td>2300</td><td></td></tr><tr><td>Glycerol Gelatin</td><td>Sigma-Aldrich</td><td>GG1</td><td></td></tr><tr><td>L-Glutamic acid</td><td>Sigma-Aldrich</td><td>G1251</td><td></td></tr><tr><td>Lithium 6-phospho-D-galactonate</td><td>Sigma-Aldrich</td><td>55962</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>methoxy-Phenazine methosulfate</td><td>Serva</td><td>28966.01</td><td></td></tr><tr><td>Nicotinamide adenine dinucleotide</td><td>Sigma-Aldrich</td><td>N0505&amp;nbsp;</td><td></td></tr><tr><td>Nicotinamide adenine dinucleotide phosphate</td><td>Sigma-Aldrich</td><td>NADP-RO</td><td></td></tr><tr><td>Nitrotetrazolium Blue chloride</td><td>Sigma-Aldrich</td><td>N6876</td><td></td></tr><tr><td>Phenazine methosulfate</td><td>Serva</td><td>32030.01</td><td></td></tr><tr><td>Polyvinyl alcohol, fully hydrolyzed</td><td>Sigma-Aldrich</td><td>P1763</td><td></td></tr><tr><td>Potassium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Merck Millipore</td><td>104873</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium L-lactate</td><td>Sigma-Aldrich</td><td>L7022</td><td></td></tr><tr><td>Bandpass filter</td><td>Edmund optics</td><td></td><td>https://www.edmundoptics.de/optics/optical-filters/bandpass-filters/Hard-Coated-OD-4-10nm-Bandpass-Filters/</td></tr><tr><td>Grey-step wedge</td><td>Stouffer Industries</td><td></td><td>http://www.stouffer.net/TransPage.htm</td></tr></tbody></table>

metabolic mapping: quantitative enzyme cytochemistry and histochemistry to determine the activity of dehydrogenases in cells and tissues

细胞代谢的改变是许多疾病的标志, 包括癌症, 心血管疾病和感染。细胞代谢运动单元为酶, 其活性在许多层次上受到严格调控, 包括转录、mRNA 稳定性、平移、后转化和功能水平。这一复杂的调控意味着常规的定量或成像检测, 如定量 mRNA 实验, 西方印迹和免疫组化, 产生不完全信息关于酶的终极活动, 它们的功能和/或它们的亚细胞定位。定量酶化学和组织化学 (即,代谢映射) 显示了关于原位酶活性及其动力学、功能和亚细胞定位的深入信息, 在一个几乎真实的情况下。
我们描述了一种检测脱氢酶活性的协议, 这些酶是执行氧化还原反应以减少辅助因子, 如 NAD (P) + 和时尚的酵素。细胞和组织切片的孵化在一个特定的酶活性的一个脱氢酶的培养基。随后, 作为调查对象的脱氢酶在其亚细胞部位进行酶活性。在反应介质的化学反应中, 这最终会在脱氢酶的活性部位产生蓝色 formazan。因此, formazan 的吸光度直接测量脱氢酶的活性, 可以用单色光镜和图像分析来量化。这个协议的数量方面使研究人员能够从这些化验中得出统计结论。
除观察研究外, 该技术还可用于特定酶的抑制研究。在这种情况下, 研究得益于新陈代谢映射的真实到自然的优势, 给予就地结果, 这可能与体外酶抑制研究相比,在生理上更相关。总之, 代谢图谱是研究细胞或组织水平代谢的不可缺少的技术。该技术易于采用, 提供了深入、全面、综合的代谢信息, 能够快速定量分析。

为了研究特定脱氢酶的活性, 我们展示了几种制备孵化培养基的规程。对于每个脱氢酶, 在 PVA 溶液中溶解所列出的试剂, 同时保持至少37摄氏度的温度。试剂应按照表中列出的顺序溶解,即nitroBT 总是先溶解, (m) PMS 总是最后溶解。每5毫克的 nitroBT 溶解在40µL 混合 (20 µL 乙醇 + 20 µL 甲基酰胺)。所有容量是每1毫升 18% PVA 溶液, 可用于染色 ~ 2 组织切片或 ~ 4 细胞制剂在玻璃幻灯片。NitroBT、NAD+、NADP+、ADP 和基板解决方案应新鲜地进行。NaN3、氯化镁2和 (m) PMS 解决方案可以存储在4°c 一个月。18% 在磷酸盐缓冲液中溶解的 PVA 可贮存在60摄氏度2周。18% PVA 溶液的 pH 值可以用0.1 米磷酸二氢钾 (即2PO4) 和 0.1 M 钠磷酸氢 (NA2HPO4) 缓冲区的不同成分来设置。显示潜伏期提供了良好的起点, 但最佳潜伏期取决于物种, 组织类型和保存的组织。一般而言, 细胞制剂的孵化时间应长于组织切片, 以获得类似的 formazan 生产水平。
野生型异柠檬酸脱氢酶1和 2 (IDH1/2) 催化异柠檬酸到αα-(αKG) 的转化, 并随NADP + 的相应减少, 分别在细胞质和线粒体中的。这些酶最近引起了兴趣, 因为IDH1/2突变发生在各种类型的人类癌症, 包括原发性脑癌 (胶质瘤) 和结直肠癌, 并提供了一个独特的机会, 以证明的可能性代谢图谱。IDH1突变禁用 IDH1 野生型酶活性, 也会诱发新酶活性, 导致 oncometabolite d-2-戊 (D-2HG) 的生产和随后积累,11这是在其他地方详细讨论。12代谢映射实验表明, 在异型IDH1突变的结直肠癌细胞中, NADP+依赖性 IDH1/2 活性明显低于IDH1野生类型 (图 1A)。这种差异是通过对单色光显微照片 (图1B) 的图像分析来量化的。12
在图 2中显示了新陈代谢映射实验的另一个例子, 这是一个恒温器的小鼠大脑的图像, 它是用人脑胶质瘤细胞注射的。IDH1/2 是人脑中最重要的上调, 其活性在野生型IDH1/2胶质母细胞瘤中有很大的作用, 而 IDH1/2 在啮齿类动物脑中的能力更低。11图 2A显示了人脑胶质瘤细胞中高 NADP+依赖性 IDH1/2 活动与正常小鼠大脑中 NADP+依赖性 IDH1/2 活动之间的差异。在图 2B中, IDH1/2 活动被量化为每分钟组织每毫升的µmol 产量。
IDH1/2 的酶反应相对简单, 但乳酸脱氢酶 (LDH) 是一种较为复杂的酶复合物。乳酸脱氢酶通过将 NAD+还原为 NADH 或反之亦然, 将乳酸的可逆转化为丙酮酸。乳酸和丙酮酸的供应以及 LDH 酶复合物的组成决定了乳酸是否主要转化为丙酮酸 (由 LDH B 和产量 NADH 催化), 或者丙酮酸酯是否主要转化为乳酸 (这是由 LDH A 催化并消耗 NADH)。13代谢映射实验能够可视化 ldh-B 反应的活性, 但它们对 ldh 的活性是 "盲目的"-反应是因为 NADH 的生产不会发生, 因此 nitroBT 不会降低到 formazan。图 3显示了在缺少基板 (图 3A) 的情况下, 在高浓度乳酸 (图 3B) 的情况下, 由 NAD+相关的 LDH 活性 (,即将乳酸转化为丙酮酸) 在人脑部分.), 在存在6毫米乳酸 (图 3C) 和在存在低浓度乳酸和较高浓度的丙酮酸 (图 3D)。这些实验的结果说明新陈代谢的映射充分地捕获了在组织切片中的 NAD+依赖的 LDH 活性取决于乳酸和丙酮酸在反应培养基中的可用性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56843/56843fig1.jpg">
图 1.NADP+依赖于人大肠癌细胞的 IDH1/2 活性。() HCT116 人大肠癌细胞染色后 NADP+依赖性 IDH1/2 活动对1-10 毫米异柠檬酸和0.8 毫米 NADP + 的显微照片的代表单色光.刻度条 = 50 µm. (B) 通过在 (A) 和图像分析中使用单色光, 显示由 NADP+相关 IDH1/2 活动 nitroBT 产生的蓝色 formazan 的吸光度的量化。缩写: IDH1重量/重量, 异柠檬酸脱氢酶1野生型;IDH1物, 异柠檬酸脱氢酶1突变。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56843/56843fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56843/56843fig2.jpg">
图 2.NADP+依赖于人类胶质瘤样本的 IDH1/2 活动。() 一个包含健康小鼠大脑 (h) 和人类胶质瘤异种瘤 (t) 的小鼠大脑部分的代表显微照片在 NADP 和依赖的 IDH1/2 活动后, 存在0。8毫米 NADP+和2毫米异柠檬酸。缩放条 = 200 µm (B) 酶活性量化的小鼠脑 (h) 和人胶质瘤异种 (t) 中显示的 (A) 使用兰伯特-啤酒定律。误差线表示标准偏差。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56843/56843fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56843/56843fig3.jpg">
图 3.与+相关的乳酸脱氢酶 (LDH) 活性的人脑标本。为NAD+依赖的 LDH 活性 (A) 对控制条件进行染色后, 对人脑样品的序列显微照片的代表性 (在缺勤基底和丙酮酸, 3 毫米 NAD +) (B)反对150毫米乳酸在缺乏丙酮酸 (C) 对6毫米乳酸在缺乏丙酮酸并且 (D) 反对6毫米乳酸在存在18毫米丙酮酸的存在下, 乳酸对丙酮酸反应的竞争产品抑制剂。缩放条 = 200 µm.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56843/56843fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>酶:</td>
			<td>G6PDH</td>
			<td>Ldh</td>
			<td>Sdh</td>
			<td>MDH</td>
			<td>IDH1</td>
			<td>IDH2</td>
			<td>IDH3</td>
			<td>6PGD</td>
			<td>粤海</td>
		</tr>
<tr>
<td>PVA 18% 在100毫米磷酸盐缓冲</td>
			<td>1毫升;pH 值 = 7。4</td>
			<td>1毫升; pH 值 = 7。4</td>
			<td>1毫升; pH 值 = 8。0</td>
			<td>1毫升;pH 值 = 7。4</td>
			<td>1毫升;pH 值 = 7。4</td>
			<td>1毫升;pH 值 = 7。4</td>
			<td>1毫升;pH 值 = 7。4</td>
			<td>1毫升;pH 值 = 8。0</td>
			<td>1毫升;pH 值 = 8。0</td>
		</tr>
<tr>
<td>5毫米 NitroBT</td>
			<td>5 mg/40 µL 混合</td>
			<td>5 mg/40 µL 混合</td>
			<td>5 mg/40 µL 混合</td>
			<td>5 mg/40 µL 混合</td>
			<td>5 mg/40 µL 混合</td>
			<td>5 mg/40 µL 混合</td>
			<td>5 mg/40 µL 混合</td>
			<td>5 mg/40 µL 混合</td>
			<td>5 mg/40 µL 混合</td>
		</tr>
<tr>
<td>5 mM 南3 </td>
			<td>10µL</td>
			<td>10µL</td>
			<td>10µL</td>
			<td>10µL</td>
			<td>10µL</td>
			<td>10µL</td>
			<td>10µL</td>
			<td>10µL</td>
			<td></td>
		</tr>
<tr>
<td>5 mM 氯化镁2</td>
			<td>10µL</td>
			<td></td>
			<td></td>
			<td>10µL</td>
			<td>10µL</td>
			<td>10µL</td>
			<td>10µL</td>
			<td>10µL</td>
			<td></td>
		</tr>
<tr>
<td>3 mM NAD+</td>
			<td></td>
			<td>10µL</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>10µL</td>
			<td></td>
			<td>10µL</td>
		</tr>
<tr>
<td>0.8 毫米 NADP</td>
			<td>10µL</td>
			<td></td>
			<td></td>
			<td>10µL</td>
			<td>10µL</td>
			<td>10µL</td>
			<td></td>
			<td>10µL</td>
			<td></td>
		</tr>
<tr>
<td>2毫米 ADP</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>10µL</td>
		</tr>
<tr>
<td>衬 底</td>
			<td>10毫米 glucose-6-phosphate</td>
			<td>150毫米乳酸</td>
			<td>50毫米琥珀酸</td>
			<td>100毫米苹果酸</td>
			<td>2毫米异柠檬酸</td>
			<td>2毫米异柠檬酸</td>
			<td>2毫米异柠檬酸</td>
			<td>10毫米 6-磷-galactonate</td>
			<td>10毫米谷氨酸</td>
		</tr>
<tr>
<td>0.32 毫米 mPMS</td>
			<td>10µL</td>
			<td>10µL</td>
			<td></td>
			<td>10µL</td>
			<td>10µL</td>
			<td></td>
			<td></td>
			<td>10µL</td>
			<td></td>
		</tr>
<tr>
<td>0.2 毫米 PMS</td>
			<td></td>
			<td></td>
			<td>10µL</td>
			<td></td>
			<td></td>
			<td>10µL</td>
			<td>10µL</td>
			<td></td>
			<td>10µL</td>
		</tr>
<tr>
<td>潜伏期</td>
			<td>5分钟</td>
			<td>30分钟</td>
			<td>60分钟</td>
			<td>60分钟</td>
			<td>30分钟</td>
			<td>30分钟</td>
			<td>30分钟</td>
			<td>10分钟</td>
			<td>30分钟</td>
		</tr>
</tbody></table>
表1。脱氢酶代谢映射协议示意图概述。缩写: ADP, 磷酸腺苷;mPMS (甲氧基)-5-酚嗪甲基硫酸盐;nitroBT, 硝基四氮唑蓝氯;G6PDH, glucose-6-phosphate 脱氢酶;乳酸脱氢酶;SDH, 琥珀酸脱氢酶;MDH, 苹果酸脱氢酶;IDH, 异柠檬酸脱氢酶;6PGD, 6-phosphogluconate 脱氢酶;粤海谷氨酸脱氢酶

Watch this Scientific Journal Video about Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues at JoVE.com

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

在这里, 我们提出了一个协议, 可用于显微镜下可视化和量化的活动脱氢酶在细胞和组织及其动力学, 功能和亚细胞定位。

代谢图谱: 定量酶化学和组织化学法测定脱氢酶在细胞和器官中的活性

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...

metabolic-mapping-quantitative-enzyme-cytochemistry-histochemistry-to

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.6K 次观看.  University of Amsterdam, Amsterdam, The Netherlands. 在这里, 我们提出了一个协议, 可用于显微镜下可视化和量化的活动脱氢酶在细胞和组织及其动力学, 功能和亚细胞定位。

视频: Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

质谱法和基于发光的-途径表征阶段我代谢能力的<em&gt;体外</em&gt;细胞培养

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

科研

生物化学

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

含黄素线粒体脱氢酶同时测定超氧化物/过氧化氢和 NADH 生产

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

环境科学

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

映射代谢: 直接在组织中监测乳酸脱氢酶活性

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

稳定同位素追踪和LC-MS分析以测量DHODH和SDH活性

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

作者聚焦：测量哺乳动物线粒体功能的氧非依赖型检测  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

测量哺乳动物线粒体功能的氧非依赖性测定

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

使用C2C12成肌细胞的生物能简介实验

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

生物学

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

使用细胞色素线粒体呼吸功能的可视化 C氧化酶/琥珀酸脱氢酶（COX / SDH），双标组织化学

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

<meta charset="utf8"></head><body>改变线粒体功能以用于治疗和生物杀虫剂应用

Unveiling Xenobiotic Transport and Effects in Isolated Mitochondria: Insights from Respirometric and Enzymatic Assays

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). However, their significance extends far beyond energy production. Dysregulation of mitochondrial bioenergetics can trigger intracellular cascades that impact health and cellular function. Given their physiological importance, there is growing interest in exploring the pharmacological potential of mitochondrial function for developing new therapies and bioproducts. Modulating mitochondrial bioenergetics could provide innovative approaches to address challenges such as neurodegenerative disorders, metabolic diseases, and cancer, as well as to develop bioproducts for controlling pests and vector-borne diseases.
This work presents a method for assessing the effects of xenobiotics on the electron transport chain (ETC) using various substrates, including glutamate and NADH for complex I, succinate for complex II, and cytochrome c (both oxidized and reduced) for complexes III and IV. This methodology allows the activation or inhibition of electron transport through mitochondrial complexes to be evaluated using a respirometer and spectrophotometer. The mitochondria can be sourced from isolated mitochondria, fragmented cells, or homogenized tissue from various species.
In the laboratory, mitochondrial function has been analyzed in Aedes aegypti and Wistar rats, but this method is also applicable to other species, such as Rhipicephalus microplus and Rhodnius prolixus. This approach provides the basis for theorizing about the existence of uncoupler proteins and species-specific oxidizable substrate preferences influenced by their unique energetic demands. Recent findings offer valuable insights into innovative bioinsecticide design strategies that target mitochondrial function, holding significant potential for effectively controlling vector-borne diseases and pest infestations.

<meta charset="utf8"></head><body>揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

This study introduces a methodology for studying xenobiotic effects on mitochondrial function via respirometric and enzymatic assays. It examines electron transport chain modulation in various species, offering insights for targeted biopesticide development against insect vector-borne diseases.

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). ...

代谢图谱: 定量酶化学和组织化学法测定脱氢酶在细胞和器官中的活性

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质谱法和基于发光的-途径表征阶段我代谢能力的

含黄素线粒体脱氢酶同时测定超氧化物/过氧化氢和 NADH 生产

检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

映射代谢: 直接在组织中监测乳酸脱氢酶活性

测量哺乳动物线粒体功能的氧非依赖性测定

测量哺乳动物线粒体功能的氧非依赖性测定

测量哺乳动物线粒体功能的氧非依赖性测定

使用C2C12成肌细胞的生物能简介实验

使用细胞色素线粒体呼吸功能的可视化 C氧化酶/琥珀酸脱氢酶（COX / SDH），双标组织化学

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解