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, 可以导致更确凿的信息, 不只是在哪里乳酸脱氢酶是现在, 但他们实际上功能。此处描述的方法可以与免疫组化相结合, 以使所选蛋白质的水平 (例如, 细胞谱系标记) 与乳酸脱氢酶活性相同的组织样本确定。与基于 MRI 的乳酸脱氢酶活性监测方法相比, 该协议提供了更大的空间分辨率, 有助于确定组织内具有高酶活性的特定细胞。.与基于荧光基片的新陈代谢方法相比, 所描述的协议检测信号不受荧光1的限制。米勒和合著者通过监测转化为 formazan7, 对五种不同酶的时间依赖性和基质依赖性进行了相似的分析。反应被发现遵循化学计量学原理的酶动力学。米勒和合著者通过与线粒体染色7的联合定位, 来分辨非线粒体酶的线粒体酶。他们使用 5-氰基-23-二 (对甲苯基) 四氮唑氯 (CTC) 作为检测试剂, 其次为亚细胞共定位分析7的共焦显微术。
所描述的方法也有缺点。虽然基于 MRI 的方法可用于活体生物, 但所描述的方法需要冷冻或新鲜的组织。冷冻组织仅适用于几个月, 因为酶的活性随着时间的推移而降低, 即使冷冻。此外, 由于基体提供过剩, 该方法提供了有关 "活动潜力" 假设基板存在。如果基底的水平是有限的, 那么观察到的活动分布可能会偏离生理上发生的活动量。此外, 如果有相同酶的不同亚型, 执行相同的酶活性, 这里提出的方法无法区分它们。例如, 所提出的方法不能区分 LDHA 与 LDHB 的活动。解决这个问题的一种方法是使用基因工程小鼠, 其中单一酶异构体是基因灭活。
我们试验了 PMS, 一个光化学法稳定的电子载体, 介导电子在 NADH 和四氮唑染料之间的转移23。虽然一些科学家报告说, pms 抑制葡萄糖 6-磷酸脱氢酶活性, 发现 pms 无益于5, 我们发现 pms 对酶的定位有价值, 并将其纳入我们的协议。还有一些作者报告使用聚乙烯醇来限制葡萄糖 6-磷酸脱氢酶1的扩散。在我们的实验中, 我们没有发现聚乙烯醇是有益的, 并消除它。
几步对染色的成功是至关重要的。一个重要的问题是确保组织样本正确放置在 cyromolds。皮肤切片应平整, 使切片切过皮肤。此外, 如果要准备多个节, 那么理想的情况是, 如果它们嵌入在同一 cryomold 中, 以便在同一张幻灯片上切片。这将有助于消除由于幻灯片的厚度与恒温器削减的变化。同样重要的是要确保组织仍然保留酶活性。
最好的时间来结束的反应应该有经验的确定。有必要在潜伏期密切监测幻灯片, 并确定适当的时间添加 counterstain 和登上幻灯片的基础上的蓝色污点的强度。如果样品的污渍太轻, 就不可能确定活动的位置。如果样品是 overstained 的, formazan 沉淀就会扩散, 使得很难确定最初处于活动高位的特定区域。
我们预计, 这项议定书将是宝贵的科学家有广泛的问题, 关于新陈代谢的作用。应用包括代谢活动和细胞谱系标记或增殖标记的共同染色, 评估不同酶的贡献与遗传模型, 评估酶活性的亚细胞定位, 监测酶活性在患病组织或发展期间。

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 border="0" cellpadding="0" cellspacing="0" style="width:815px;" width="814">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:312px;">Surgical instruments</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;">For collecting skin from euthanized mice</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Tissue-tek cryomold 25 mm x 20 mm x 5 mm</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">NC9511236</td>
			<td style="width:217px;">For freezing mouse skin</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">NC9638938</td>
			<td style="width:217px;">For mounting cryomolds</td>
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			<td height="21" style="height:21px;width:312px;">Ice bucket</td>
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			<td style="width:121px;">07-210-106</td>
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			<td height="21" style="height:21px;width:312px;">Dry Ice</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Polysine Adhesion Slide</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">12-545-78</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">4% formalin</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">23-245-684</td>
			<td style="width:217px;">Dilluted in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">phosphate buffered saline, pH 7.4</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">vortex</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Tris base</td>
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			<td style="width:217px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:312px;">NAD</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:121px;">N7004</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Phenazine methosulfate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:121px;">P9625</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:312px;">Nitrotetrazolium blue chloride</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:121px;">N6876</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lithium L-lactate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:121px;">L2250</td>
			<td style="width:217px;">Substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">37&#176;C incubator (or tissue culture incubator)</td>
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			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Braziliant! Counter stain</td>
			<td style="width:164px;">Anatech</td>
			<td style="width:121px;">861</td>
			<td style="width:217px;">Counter stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Mounting medium</td>
			<td style="width:164px;">Vector Laboratories</td>
			<td style="width:121px;">H-5000&#160;</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Cover slips for slides</td>
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			<td style="width:121px;">12-544D</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Light microscope</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></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, 毛囊干细胞具有较高的乳酸脱氢酶活性, 我们扩大了对培养胚胎干细胞的研究。单独或在胚胎干细胞存在下, 对小鼠胚胎成纤维细胞作为饲养层进行分析。为分析培养细胞, 对培养基进行了吸气, 并对细胞进行了简单的固定, 并与乳酸脱氢酶染色溶液一起孵化。与小鼠胚胎成纤维细胞相比, 我们观察到胚胎干细胞中乳酸脱氢酶活性较高 (图 4)。
我们还将染色协议应用于新鲜分离的组织。小鼠依次被解剖切开。这些血管被打开和固定, 以便依次的内腔可以进入。样品用汉克的平衡盐溶液孵化, 含0.5% 卫10分钟, 然后用乳酸脱氢酶染色液10分钟乳酸脱氢酶染色液用于新鲜组织。孵化后, 图像收集, 显示在平滑肌细胞染色 (图 5)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57760/57760fig1.jpg"> 图 1: 乳酸脱氢酶的化学活性及其检测.乳酸脱氢酶转化乳酸 + NAD 为丙酮酸 + NADH + H+。产生的质子将减少硝基四氮唑到 formazan, 这将形成一个蓝色沉淀。<a href="/files/ftp_upload/57760/57760fig1large.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/57760/57760fig2.jpg"> 图 2: 在 cryomolds 中定位小鼠皮肤的示意图.鼠标皮肤应在 cryomolds 内, 使每个切口部分将包含整个皮肤从表皮, 真皮, 皮下组织和肌肉的横断面。<a href="/files/ftp_upload/57760/57760fig2large.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/57760/57760fig3.jpg"> 图 3: 小鼠皮肤乳酸脱氢酶染色显示毛囊干细胞活性高.小鼠皮肤被冷冻在 cryomolds 和孵化乳酸脱氢酶染色溶液含有乳酸。乳酸脱氢酶活性在毛囊干细胞中突出。(A、B)两种小鼠皮肤乳酸脱氢酶活性测定的图像。(C、D)两个图像的控制染色与基质乳酸隐瞒。刻度条 = 50 µm.<a href="/files/ftp_upload/57760/57760fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57760/57760fig4.jpg"> 图 4: 培养的小鼠胚胎干细胞中乳酸脱氢酶活性高.对经辐照小鼠胚胎成纤维细胞饲养层培养的小鼠胚胎干细胞进行乳酸脱氢酶活性分析。与成纤维细胞相比, 在干细胞中观察到高活性。小鼠胚胎成纤维细胞表现出相似的染色和不照射水平。图像是以20X 的目标拍摄的。鳞片棒 = 50 µm (A、B、D、E) 小鼠胚胎成纤维细胞和干细胞。(C、F)小鼠胚胎成纤维细胞单独。(丙)图像是在7分钟的孵化后采取染色溶液。(D F)图像是在20分钟的孵化后采取染色溶液。<a href="/files/ftp_upload/57760/57760fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57760/57760fig5.jpg"> 图 5: 小鼠主动脉周围平滑肌细胞高乳酸脱氢酶活性.依次与安乐死小鼠分离。血管在2% 多聚甲醛固定2分钟, 在1x 磷酸盐缓冲盐水中洗涤 3 x 10 分钟。用汉克的平衡盐溶液与0.5% 的海卫 x 和 3 x 5 分钟在磷酸盐缓冲盐水中冲洗了血管。添加乳酸脱氢酶染色液, 在37°c 培养10分钟的样品, 用1x 磷酸盐缓冲盐水冲洗 3 x 5 分钟。样品在2% 多聚甲醛被修理了1小时, 洗涤了 3 x 5 分钟与1x 磷酸盐缓冲盐水和映像。乳酸脱氢酶溶液, 含乳酸。(B) 乳酸脱氢酶溶液, 无乳酸作为阴性对照。对于每一个部分, 在左边有一个较低的放大图像, 并且指定的区域在右边显示更高的放大倍数。(A、B)全乳酸脱氢酶溶液中含有乳酸, 用于染色。B 是图像 a 中指定区域的放大图像 a. (C、D) 乳酸脱氢酶溶液无乳酸用于染色作为阴性对照。D 是 C 中指定区域的放大图像。a 和 C 被采取了以20X 目标 (鳞片酒吧 = 50 µm) 和 B 和 D 被采取了以40X 目标 (标尺 = 20 µm)。<a href="/files/ftp_upload/57760/57760fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.3K 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

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

科研

生物化学

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

用氢-氘交换质谱法定义 Hsp33's 氧化还原调节陪护活动和 Hsp33 映射构象变化

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

SUMO 结合实体 （SUBEs） 作为肝癌中 SUMO 蛋白酶的浓缩、分离、识别和表征的工具

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

通过反相液相-质谱法对无细胞蛋白合成代谢的绝对定量

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

大鼠胰腺组织快速、经济高效的RNA提取

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of &#945;1,4-linked glucose residues with branches introduced through the addition of &#945;1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

用于研究真核糖原代谢的分光光度法

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains ...