Name: mapping metabolism: monitoring lactate dehydrogenase activity directly in tissue
Uploaded: 2018-06-21T14:00:00.000+00:00
Duration: 6 min 18 s
Description: Watch this Scientific Journal Video about Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue at JoVE.com

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>

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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

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

超极化<sup&gt; 13</sup&gt;ç代谢磁共振波谱与成像

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

科研

癌症研究

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

免疫与感染

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 ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

神经科学

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, pro-inflammatory (i.e., M1-like) monocytes/macrophages display more reliance on anaerobic glycolysis and less reliance on mitochondrial oxidative phosphorylation, whereas anti-inflammatory (M2-like) macrophages display more reliance on glucose and fatty acid oxidation in the mitochondria. Here, we describe in-depth protocols for extracting macrophages from the two major monocyte/macrophage reservoirs in the body, the spleen and bone marrow, as well as injured tissues such as the heart following myocardial infarction.
Macrophages or monocytes are extracted by immunomagnetic sorting by using antibody-tagged microbeads, which easily bind to cells without compromising their phenotypes. The extracted cells are then cultured in 96-well plates, followed by extracellular flux analysis using a metabolic flux analyzer. Both glycolysis and mitochondrial oxidative phosphorylation can be measured simultaneously in small numbers of cells (as little as 2-3 &#215; 105 cells). This method can easily be performed in 1 day and produces reliable and repeatable results. Ultimately, these methods help to enhance our understanding of metabolic changes during immune and inflammatory responses to injury and disease, which could lead to the development of novel therapeutic targets for immunometabolic pathways.

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

分析脾和心脏巨噬细胞以及骨髓单核细胞中的 离体 代谢通量

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, ...

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 ...

生物学

使用 FRET 传感器监测 果蝇 中的脑代谢物运输

Visualizing Monocarboxylates and Other Relevant Metabolites in the Ex Vivo Drosophila Larval Brain Using Genetically Encoded Sensors

The high energy requirements of brains due to electrical activity are one of their most distinguishing features. These requirements are met by the production of ATP from glucose and its metabolites, such as the monocarboxylates lactate and pyruvate. It is still unclear how this process is regulated or who the key players are, particularly in Drosophila.
Using genetically encoded F&#246;rster resonance energy transfer-based sensors, we present a simple method for measuring the transport of monocarboxylates and glucose in glial cells and neurons in an ex-vivo Drosophila larval brain preparation. The protocol describes how to dissect and adhere a larval brain expressing one of the sensors to a glass coverslip. 
We present the results of an entire experiment in which lactate transport was measured in larval brains by knocking down previously identified monocarboxylate transporters in glial cells. Furthermore, we demonstrate how to rapidly increase neuronal activity and track metabolite changes in the active brain. The described method, which provides all necessary information, can be used to analyze other Drosophila living tissues.

作者聚焦:使用 果蝇 模型的脑能量代谢研究进展

Here we present a protocol to visualize the transport of monocarboxylates, glucose, and ATP in glial cells and neurons using genetically encoded F&#246;rster resonance energy transfer-based sensors in an ex-vivo Drosophila larval brain preparation.

使用基因编码传感器在 离体果蝇 幼虫大脑中可视化单羧酸盐和其他相关代谢物

The high energy requirements of brains due to electrical activity are one of their most distinguishing features. These requirements are met by the ...

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 ...

映射代谢: 直接在组织中监测乳酸脱氢酶活性

摘要

探索更多视频

此视频中的章节

超极化

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

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

果蝇幼虫和成年脑的代谢分析

分析脾和心脏巨噬细胞以及骨髓单核细胞中的离体代谢通量

测量外产酸和分析，以确定糖酵解率

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物

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

映射代谢: 直接在组织中监测乳酸脱氢酶活性

摘要

探索更多视频

此视频中的章节

超极化

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

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

果蝇幼虫和成年脑的代谢分析

分析脾和心脏巨噬细胞以及骨髓单核细胞中的 离体 代谢通量

测量外产酸和分析，以确定糖酵解率

使用基因编码传感器在 离体果蝇 幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用基因编码传感器在 离体果蝇 幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用基因编码传感器在 离体果蝇 幼虫大脑中可视化单羧酸盐和其他相关代谢物

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

分析脾和心脏巨噬细胞以及骨髓单核细胞中的离体代谢通量

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物