文森特卡塞尔是丽塔. 艾伦基金会 (http://www.ritaallenfoundation.org) 的弥尔顿 e。这项工作由国家普通医学研究所 R01 GM081686、R01 AR070245、国立医学科学院 R01 GM0866465、Eli & 伊蒂丝广泛的再生医学 & 干细胞研究中心的赠款资助 (玫瑰丘陵和哈尔 Gaba 奖), 虹膜康托妇女健康中心/加州大学洛杉矶分校 CTSI NIH 补助金 UL1TR000124, 白血病淋巴瘤协会, 冲击奖从琼森综合癌症中心到和醋酸。在这份出版物中报告的研究得到了国家卫生研究院国家癌症研究所的支持, P50CA092131 奖号。资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有任何作用。醋酸是伊莱 & 伊蒂丝广泛的再生医学 & 干细胞研究中心的成员, 加州大学洛杉矶分校分子生物学研究所, 加州大学洛杉矶分校生物信息学部门计划。

所描述的所有实验都是由洛杉矶加利福尼亚大学动物保育委员会批准的。
1. 生成冷冻鼠标皮肤的幻灯片
<ol><li>弄死小鼠按照制度政策。 注意: 遵循保护服装的制度政策。所有涉及动物的议定书都必须得到机构动物保育委员会的批准。</li>
	<li>用动物发型剪掉老鼠的头发。</li>
	<li>用剪刀在皮肤上做切口。使用镊子, 把皮肤从老鼠身上抬开。用剪刀剪掉皮肤部分。</li>
	<li>用冷冻试剂化合物填充 cryomold (见材料表)。</li>
	<li>使用针鼻钳将皮肤部分放入填充 cryomolds。东方皮肤, 使组织切片将产生一个横断面的皮肤从表皮到真皮, 皮下组织和肌肉 (图 2)。 注意: 如果可能的话, 将实验和控制鼠标放在同一 cryomold 中, 这样它们就可以被切片到同一张幻灯片上并一起处理。注意不要制造气泡。</li>
	<li>把 cryomold 放在冰桶里一块干冰的平坦表面上, 让它结冰。继续观察皮肤的方向。必要时进行调整。 注: 处理干冰时应佩戴低温手套。</li>
	<li>将 cryomolds 转移到-80 摄氏度冷藏库中存放。样品可以在冷冻库中保存约3月, 而不会造成大量的酶活性损失。不要让冰冻切片干燥。</li>
	<li>使用恒温器在-20 °c, 切片组织创建 7-10 µm 厚为最佳皮肤形态学21。</li>
</ol>2. 准备用于染色的幻灯片
<ol><li>建立要测试的幻灯片集。包括一个控制滑块, 它将被扣留, 另一张控制滑块将被扣留, 乳酸基。包括先前调查过的冰冻组织块中的阳性对照滑动。</li>
	<li>在幻灯片上, 或者在处理多张幻灯片时, 通过将幻灯片浸入包含4% 福尔马林的容器中, 将包含4% 福尔马林的皮肤部分的幻灯片简单地固定为5分钟. 吹打1毫升4% 福尔马林。 注意: 该固定将确保皮肤不会剥离从幻灯片在下列过程中, 保持皮肤形态学。 注意: 福尔马林是一种致癌物, 具有危险性;戴上手套, 不要让它接触你的皮肤, 并在化学油烟机上执行这一步骤。</li>
	<li>用至少1毫升磷酸盐缓冲盐水, pH 值为 7.4, 每张幻灯片用吹打或浸洗的幻灯片。</li>
</ol>3. 准备染色液
<ol><li>漩涡联合试剂的乳酸脱氢酶活性染色 (50 毫米三 pH 值 7.4, 750 µM NADP, 80 µM 吩 methosulfate (PMS), 600 µM nitrotetrazolium 蓝氯, 30 毫米乳酸) 在适当大小的管取决于染色量解决方案要求。 注: 1 毫升足以完全覆盖一张幻灯片。</li>
	<li>准备第二个解决方案, 其中所有试剂都存在, 除了 NAD。准备第三个溶液, 除乳酸外, 所有试剂都存在。</li>
</ol>4. 在染色液中孵化幻灯片
<ol><li>将染色液或控制溶液轻轻地吸管覆盖整个切片的准备好的幻灯片上 (1 毫升足以用于一张幻灯片)。如果将多个幻灯片一起处理, 请同时将所有幻灯片浸入染色液中 (确保容器有足够的解决方案来完全覆盖组织部分)。</li>
	<li>在一个湿润的环境中孵化的幻灯片在37摄氏度在黑暗中。如果染色液位于幻灯片的顶端, 请将幻灯片放在加湿的环境中以防止蒸发。</li>
</ol>5. 监控幻灯片
<ol><li>通过目视检查, 监测染色液从透明到蓝色的转换。当样品达到所需的蓝色水平时, 通过去除染色溶液来停止反应。 注意: 对于皮肤, 10 分钟是足够的。时间将取决于器官和酶的活动。</li>
	<li>用磷酸盐缓冲盐水冲洗吹打 (每张幻灯片都足够1毫升) 或浸泡。 注: 任何将被比较的样品必须在染色溶液中保持相同的时间。</li>
</ol>6. Counterstain 和芒特
<ol><li>当幻灯片达到适当的蓝色水平时, 吸管 counterstain 到幻灯片上。 注: Counterstains, 使核红色或绿色将提供良好的对比, 蓝色创建的 formazan 沉淀。</li>
	<li>安装有水或非水安装介质22。一个到三滴 (40-50 µL) 的安装介质是足够的, 取决于大小的封面滑动。</li>
</ol>7. 图像幻灯片和量化
<ol><li>在光显微镜下的图像幻灯片。在10X、20X 和40X 放大倍数上拍摄实验和控制样本以及所有负控制的照片。</li>
	<li>用图像分析软件确定不同区域的蓝斑强度。</li>
</ol>

<ol>
	<li>Boonacker, E., 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=Enzyme+cytochemical+techniques+for+metabolic+mapping+in+living+cells,+with+special+reference+to+proteolysis.">Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis.</a> J Histochem Cytochem. 49, 1473-1486 (2001).</li><li>Seidler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tetrazolium-formazan+system:+Design+and+histochemistry.">The tetrazolium-formazan system: Design and histochemistry.</a> Prog Histochem Cytochem. 24, 1-86 (1991).</li><li>Van Noorden, C. J. F., Frederiks, W. M. <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 Histochemistry: A Laboratory Manual of Current Methods. , (1992).</li><li>Winzer, K., Van Noorden, C. J., Kohler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+cytochemical+analysis+of+glucose-6-phosphate+dehydrogenase+activity+in+living+isolated+hepatocytes+of+European+flounder+for+rapid+analysis+of+xenobiotic+effects.">Quantitative cytochemical analysis of glucose-6-phosphate dehydrogenase activity in living isolated hepatocytes of European flounder for rapid analysis of xenobiotic effects.</a> J Histochem Cytochem. 49, 1025-1032 (2001).</li><li>Negi, D. S., Stephens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+the+histochemical+localization+of+glucose-6-phoshate+dehydrogenase+in+animal+and+plant+tissues.">An improved method for the histochemical localization of glucose-6-phoshate dehydrogenase in animal and plant tissues.</a> J Histochem Cytochem. 25, 149-154 (1977).</li><li>Boren, 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=In+situ+localization+of+transketolase+activity+in+epithelial+cells+of+different+rat+tissues+and+subcellularly+in+liver+parenchymal+cells.">In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells.</a> J Histochem Cytochem. 54, 191-199 (2006).</li><li>Miller, 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=Exploring+metabolic+configurations+of+single+cells+within+complex+tissue+microenvironments.">Exploring metabolic configurations of single cells within complex tissue microenvironments.</a> Cell Metab. , (2017).</li><li>Shen, 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=Prognostic+value+of+serum+lactate+dehydrogenase+in+renal+cell+carcinoma:+a+systematic+review+and+meta-analysis.">Prognostic value of serum lactate dehydrogenase in renal cell carcinoma: a systematic review and meta-analysis.</a> PLoS One. 11, e0166482 (2016).</li><li>Zhang, X., 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=Prognostic+significance+of+serum+LDH+in+small+cell+lung+cancer:+A+systematic+review+with+meta-analysis.">Prognostic significance of serum LDH in small cell lung cancer: A systematic review with meta-analysis.</a> Cancer Biomark. 16, 415-423 (2016).</li><li>Petrelli, F., 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=Prognostic+role+of+lactate+dehydrogenase+in+solid+tumors:+a+systematic+review+and+meta-analysis+of+76+studies.">Prognostic role of lactate dehydrogenase in solid tumors: a systematic review and meta-analysis of 76 studies.</a> Acta Oncol. 54, 961-970 (2015).</li><li>Valvona, C. J., Fillmore, H. L., Nunn, P. B., Pilkington, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+and+function+of+lactate+dehydrogenase+A:+Therapeutic+potential+in+brain+tumor.">The regulation and function of lactate dehydrogenase A: Therapeutic potential in brain tumor.</a> Brain Pathol. 26, 3-17 (2016).</li><li>Markert, C. L., Shaklee, J. B., Whitt, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+a+gene:+Multiple+genes+for+LDH+isozymes+provide+a+model+of+the+evolution+of+gene+structure,+function+and+regulation.">Evolution of a gene: Multiple genes for LDH isozymes provide a model of the evolution of gene structure, function and regulation.</a> Science. 189, 102-114 (1975).</li><li>Read, J. A., Winter, V. J., Eszes, C. M., Sessions, R. B., Brady, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+altered+activity+of+M-+and+H-isozyme+forms+of+human+lactate+dehydrogenase.">Structural basis for altered activity of M- and H-isozyme forms of human lactate dehydrogenase.</a> Proteins. 43, 175-185 (2001).</li><li>Holness, M. J., Sugden, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pyruvate+dehydrogenase+complex+activity+by+reversible+phosphorylation.">Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation.</a> Biochem Soc Trans. 31, 1143-1151 (2003).</li><li>Yi, W., 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=Phosphofructokinase+1+glycosylation+regulates+cell+growth+and+metabolism.">Phosphofructokinase 1 glycosylation regulates cell growth and metabolism.</a> Science. 337, 975-980 (2012).</li><li>Fan, 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=Tyrosine+phosphorylation+of+lactate+dehydrogenase+A+is+important+for+NADH/NAD(+)+redox+homeostasis+in+cancer+cells.">Tyrosine phosphorylation of lactate dehydrogenase A is important for NADH/NAD(+) redox homeostasis in cancer cells.</a> Mol Cell Biol. 31, 4938-4950 (2011).</li><li>Zhao, D., 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=Lysine-5+acetylation+negatively+regulates+lactate+dehydrogenase+A+and+is+decreased+in+pancreatic+cancer.">Lysine-5 acetylation negatively regulates lactate dehydrogenase A and is decreased in pancreatic cancer.</a> Cancer Cell. 23, 464-476 (2013).</li><li>Vanderlinde, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+total+lactate+dehydrogenase+activity.">Measurement of total lactate dehydrogenase activity.</a> Ann Clin Lab Sci. 15, 13-31 (1985).</li><li>Nelson, S. 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=Metabolic+imaging+of+patients+with+prostate+cancer+using+hyperpolarized+[1-(1)(3)C]pyruvate.">Metabolic imaging of patients with prostate cancer using hyperpolarized [1-(1)(3)C]pyruvate.</a> Sci Transl Med. 5, 198ra108 (2013).</li><li>Flores, 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=Lactate+dehydrogenase+activity+drives+hair+follicle+stem+cell+activation.">Lactate dehydrogenase activity drives hair follicle stem cell activation.</a> Nat Cell Biol. , (2017).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+tissues.">Cryosectioning tissues.</a> CSH Protoc. 2008, (2008).</li><li>Espada, 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=Non-aqueous+permanent+mounting+for+immunofluorescence+microscopy.">Non-aqueous permanent mounting for immunofluorescence microscopy.</a> Histochem Cell Biol. 123, 329-334 (2005).</li><li>Hisada, R., Yagi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1-Methoxy-5-methylphenazinium+methyl+sulfate.+A+photochemically+stable+electron+mediator+between+NADH+and+various+electron+acceptors.">1-Methoxy-5-methylphenazinium methyl sulfate. A photochemically stable electron mediator between NADH and various electron acceptors.</a> J Biochem. 82, 1469-1473 (1977).</li></ol>

作者没有相互竞争的利益来披露。

这里描述的方法可以用来监测乳酸脱氢酶或其他代谢酶的活性, 产生 NADH 或, 在不同的细胞类型组织内或组织内的不同部分, 随着时间的推移。乳酸脱氢酶是了解干细胞和肿瘤生物学的重要酶, 在单个细胞内监测乳酸脱氢酶活性的能力很可能对这种酶的功能有重要的洞察力。
与其他方法相比, 本议定书的一个重要优点是, 监测酶活性的能力, 而不仅仅是蛋白质丰度, 可以导致更确?...

了解组织内酶具有高或低活性的部位, 对于理解发育和生理学是必不可少的。转录或蛋白质水平经常被用作酶活性的替代。虽然这类研究可以提供信息, 但它们并不能为确定酶的活性 (如平移后的修饰、蛋白质复合物的存在或酶的亚细胞定位) 的关键。当酶活性被直接测量, 它经常被监测在匀质蛋白裂解物, 不再包含有关单个细胞的信息, 或在组织内的高或低活动细胞的空间分布。
我们提供了一个详细的协议, 用于映射组织样本中酶活性的空间分布。该方法的基础是早期的研究表明, 四氮唑盐可用于本地化的活动, 脱氢酶, 硝酸和氧化酶在冷冻组织1。利用这些方法, 当质子转移到四氮唑盐2、3时, 就形成了水不溶 formazan。Glucose-6-phosphate 脱氢酶产生的四氮唑和质子, 并已检测到的活动。Glucose-6-phosphate 脱氢酶已监测在欧洲比目鱼肝细胞4, 在肺泡型2细胞的肺5和肾单位的肾脏5。四氮唑盐也被用来监测冷冻组织6中的 transketolase 活性。最近还使用类似的方法来监测相邻幻灯片7上同一组织中多个脱氢酶的活动。
这里我们描述了一种使用四氮唑盐监测乳酸脱氢酶活性的空间分布的方法 (图 1)。乳酸脱氢酶可将糖酵解生成的丙酮酸转化为乳酸, 并逆转反应。乳酸脱氢酶活性是丙酮酸盐进入 tricarboxylic 酸循环的重要决定因素, 与乳酸的分泌有关。血液中的乳酸水平经常被用来诊断一系列疾病, 包括癌症8、9、10, 因为它可以表明疾病或伤害已经损坏的细胞和酶已经释放。
乳酸脱氢酶基因有四: LDHA、LDHB、LDHC 和 LDHD11。LDHA 和 LDHB 被认为是来自早期 LDHA 基因12的重复。LDH 是活跃的聚丙烯和 LDHA 和 LDHB 可以形成 homotetramers 和 heterotetramers 彼此。据报道, LDHA 对丙酮酸有较高的亲和力, 而 LDHB 对乳酸有较高的亲和力, 并优先将乳酸转化为丙酮酸13。LDHA 启动子包含 HIF1α、cMYC 和 FOXM1 转录因子11的绑定站点。此外, 像许多其他酵酶14,15, LDH 可以修改后, 翻译修改。成纤维细胞生长因子受体1可以 phosphorylate LDHA 在 Y10, 促进聚丙烯形成, 或 Y83, 促进 NADH 基质结合16。LDHA 也可以乙酰17。由于这些原因, 对 ldh 活性的完全了解不仅需要监测 ldh 蛋白水平, 还要监控酶的活性。
除了我们在这里提出的方法外, 还使用了其他方法来监测乳酸脱氢酶的活性。乳酸脱氢酶活性可以监测 spectrophotometrically 在匀质蛋白裂解。NADH 乳酸转化为丙酮酸的世代可以根据 340 nm 的吸光度来测定, 而 NADH 的消失可以监测, 因为丙酮酸转化为乳酸18。乳酸脱氢酶活性也受到磁共振成像 (MRI) 监测。13丙酮酸盐可以被管理, 丙酮酸转化为乳酸可以被监测为 [1-13c] 乳酸/[1-13c] 丙酮酸的比例。1-13c] 乳酸/[1-13c] 丙酮酸盐的高比在癌症组织19被观察了。虽然 MRI 的方法可以提供关于正常和疾病组织中乳酸脱氢酶活性的信息, 但方法没有确定特定细胞活动水平所需的分辨率。这里提供的方法可以提供关于在组织区域甚至在单个细胞中乳酸脱氢酶活性的信息。
应用原位活性测定法, 发现在小鼠皮肤毛囊干细胞中乳酸脱氢酶活性高20。我们还利用该方法对培养的胚胎干细胞中乳酸脱氢酶的活性进行了监测, 发现干细胞的活性高于饲养层。最后, 对新鲜小鼠主动脉中乳酸脱氢酶的活性进行了监测, 观察了平滑肌细胞的染色。这里我们描述了一个详细的协议, 以监测冷冻小鼠皮肤乳酸脱氢酶活性。

<table><tbody><tr><td>Surgical instruments</td><td></td><td></td><td>For collecting skin from euthanized mice</td></tr><tr><td>Tissue-tek cryomold 25 mm x 20 mm x 5 mm</td><td>Fisher Scientific</td><td>NC9511236</td><td>For freezing mouse skin</td></tr><tr><td>Tissue-Tek O.C.T. compound</td><td>Fisher Scientific</td><td>NC9638938</td><td>For mounting cryomolds</td></tr><tr><td>Ice bucket</td><td>Fisher Scientific</td><td>07-210-106</td><td></td></tr><tr><td>Dry Ice</td><td></td><td></td><td></td></tr><tr><td>Polysine Adhesion Slide</td><td>Fisher Scientific</td><td>12-545-78</td><td></td></tr><tr><td>4% formalin</td><td>Fisher Scientific</td><td>23-245-684</td><td>Dilluted in water</td></tr><tr><td>phosphate buffered saline, pH 7.4</td><td></td><td></td><td></td></tr><tr><td>vortex</td><td></td><td></td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>23-245-684</td><td></td></tr><tr><td>NAD</td><td>Sigma-Aldrich</td><td>N7004</td><td></td></tr><tr><td>Phenazine methosulfate</td><td>Sigma-Aldrich</td><td>P9625</td><td></td></tr><tr><td>Nitrotetrazolium blue chloride</td><td>Sigma-Aldrich</td><td>N6876</td><td></td></tr><tr><td>Lithium L-lactate</td><td>Sigma-Aldrich</td><td>L2250</td><td>Substrate</td></tr><tr><td>37&amp;deg;C incubator (or tissue culture incubator)</td><td></td><td></td><td></td></tr><tr><td>Braziliant! Counter stain</td><td>Anatech</td><td>861</td><td>Counter stain</td></tr><tr><td>Mounting medium</td><td>Vector Laboratories</td><td>H-5000&amp;nbsp;</td><td></td></tr><tr><td>Cover slips for slides</td><td>Fisher Scientific</td><td>12-544D</td><td></td></tr><tr><td>Light microscope</td><td></td><td></td><td></td></tr></tbody></table>

mapping metabolism: monitoring lactate dehydrogenase activity directly in tissue

在空间和时间内绘制酶活性是理解正常组织和疾病细胞行为的分子基础的关键。原位代谢活性测定可以提供有关组织内代谢活动的空间分布的信息。我们提供了一个详细的协议, 以监测乳酸脱氢酶的活性直接在组织样品。乳酸脱氢酶是一种重要的决定因素, 即食用葡萄糖是否会通过有氧或厌氧糖酵解转化为能量。含有乳酸和 NAD 的溶液被提供给冰冻组织切片。乳酸脱氢酶活性高的细胞将提供的乳酸转化为丙酮酸, 同时将所提供的烟酰胺腺嘌呤二核苷酸 (NAD) 转化为 NADH 和质子, 可根据还原nitrotetrazolium 蓝色到 formazan, 这是可视化为蓝色沉淀。我们描述了一个详细的协议, 以监测乳酸脱氢酶活性的小鼠皮肤。应用该协议, 我们发现乳酸脱氢酶活性是高的静态毛囊干细胞在皮肤内。将该协议应用于培养的小鼠胚胎干细胞, 发现培养的胚胎干细胞比小鼠胚胎成纤维细胞有更高的染色。对刚分离的小鼠主动脉的分析显示, 垂直于主动脉的平滑肌细胞染色。所提供的方法可用于空间地映射在冷冻或新鲜组织中产生质子的酶活性。

我们以前报告的结果,在现场活性测定在老鼠皮肤20。如图 3所示, 我们观察到当上述程序被遵循时, 在毛囊底部毛囊干细胞中乳酸脱氢酶活性较高。这些发现通过荧光活化细胞分选皮肤的毛囊干细胞和确认高乳酸脱氢酶活性的干细胞室与活性检测的排序细胞。
根据我们的发现, 毛囊...

Watch this Scientific Journal Video about Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue at JoVE.com

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

映射代谢: 直接在组织中监测乳酸脱氢酶活性

我们描述了一种用于映射酶活性的空间分布的协议, 它能直接在组织样品中生成 nicotinatmide 腺嘌呤二核苷酸磷酸 (NAD (P) h) + h。

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

mapping-metabolism-monitoring-lactate-dehydrogenase-activity-directly

Research

JoVE Journal

Biochemistry

11.4K 次观看.  UCLA. 我们描述了一种用于映射酶活性的空间分布的协议, 它能直接在组织样品中生成 nicotinatmide 腺嘌呤二核苷酸磷酸 (NAD (P) h) + h。

视频: Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

复透皮电化学传感的空心微针为基础的传感器

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

科研

生物工程

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

荷尔斯坦奶牛肝线粒体耗氧量和质子泄漏动力学评价线粒体呼吸

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

生物化学

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

评估从3D多细胞肺肿瘤球体中分离的癌症相关成纤维细胞的线粒体健康

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...

癌症研究

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

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation.
We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase&#39;s activity. The formazan&#39;s absorbance is therefore a direct measure of the dehydrogenase&#39;s activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays.
Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Here, we present a protocol that can be used to microscopically visualize and quantify activity of dehydrogenases in cells and tissues and its kinetics, function and subcellular localization.

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 ...

免疫与感染

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

制备胰腺腺泡细胞钙成像的目的，细胞损伤的测量，以及腺病毒感染

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

生物学

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

实验教学中乳糖酶活性的测定

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

乳酸脱氢酶螯合测定--一种简便、可靠的测定哺乳动物细胞中自噬性螯合活性的方法

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Evaluating Cytotoxicity and Membrane Integrity in Lung Slices via Lactate Dehydrogenase Assay

Source: Neuhaus, V., et al. <a href="https://app.jove.com/v/57042">Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices.</a> J. Vis. Exp. (2018).
This video demonstrates the lactate dehydrogenase, LDH assay to measure the cytotoxic effect of a test substance and its effect on the membrane integrity in human precision-cut lung slices. The assay provides a quantitative measure of cell death or damage in the sample.

通过乳酸脱氢酶测定评估肺切片中的细胞毒性和膜完整性

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. General Preparation of Human PCLS ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells

Source: Luhr, M. et al., <a href="https://app.jove.com/v/57971">The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells.</a> J. Vis. Exp. (2018)
The video demonstrates an assay for assessing bulk autophagic sequestration activity in mammalian cells. It utilizes electroporation to release cytosolic proteins, including lactate dehydrogenase (LDH). The sequestered LDH and total cellular LDH fractions are determined to assess LDH sequestration, providing insights into the overall bulk sequestration activity.

定量哺乳动物细胞中的大量自噬隔离活性

1. Cell Seeding and Treatment

	Culture adherent cells in 75 cm2&#160;tissue culture flasks in a humidified incubator with 5% CO2&#160;at 37 &#176;C, ...