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

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

<ol>
	<li>Pavlova, N. N., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Emerging+Hallmarks+of+Cancer+Metabolism.">The Emerging Hallmarks of Cancer Metabolism.</a> Cell Metab. 23 (1), 27-47 (2016).</li><li>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=Imaging+enzymes+at+work:+metabolic+mapping+by+enzyme+histochemistry.">Imaging enzymes at work: metabolic mapping by enzyme histochemistry.</a> J Histochem Cytochem. 58 (6), 481-497 (2010).</li><li>Van Noorden, C. J. F., Mcmanus, L. M., Mitchell, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathobiology of Human Disease. , 3760-3774 (2014).</li><li>Chieco, P., Jonker, A., De Boer, B. A., Ruijter, J. M., 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=Image+cytometry:+protocols+for+2D+and+3D+quantification+in+microscopic+images.">Image cytometry: protocols for 2D and 3D quantification in microscopic images.</a> Prog Histochem Cytochem. 47 (4), 211-333 (2013).</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>Van Noorden, C. J. F., Butcher, R. G., Stoward, P. J., Pearse, A. G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histochemistry, theoretical and applied. Vol 3.: enzyme histochemistry. , 355-432 (1991).</li><li>Van Noorden, C. J., Tas, J., Vogels, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytophotometry+of+glucose-6-phosphate+dehydrogenase+activity+in+individual+cells.">Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells.</a> Histochem J. 15 (6), 583-599 (1983).</li><li>Butcher, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+in+tissue+sections+of+the+two+formazans+derived+from+nitroblue+tetrazolium+in+dehydrogenase+reactions.">The measurement in tissue sections of the two formazans derived from nitroblue tetrazolium in dehydrogenase reactions.</a> Histochem J. 10 (6), 739-744 (1978).</li><li>Matsumoto, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell biological applications of confocal microscopy. , (2002).</li><li>Errington, R. J., White, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+dynamic+cell+volume+in+situ+by+confocal+microscopy.">Measuring dynamic cell volume in situ by confocal microscopy.</a> Methods Mol Biol. 122, 315-340 (1999).</li><li>Molenaar, R. J., Radivoyevitch, T., Maciejewski, J. P., van Noorden, C. J., Bleeker, F. 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+driver+and+passenger+effects+of+isocitrate+dehydrogenase+1+and+2+mutations+in+oncogenesis+and+survival+prolongation.">The driver and passenger effects of isocitrate dehydrogenase 1 and 2 mutations in oncogenesis and survival prolongation.</a> Biochim Biophys Acta. 1846 (2), 326-341 (2014).</li><li>Molenaar, R. 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=Radioprotection+of+IDH1-Mutated+Cancer+Cells+by+the+IDH1-Mutant+Inhibitor+AGI-5198.">Radioprotection of IDH1-Mutated Cancer Cells by the IDH1-Mutant Inhibitor AGI-5198.</a> Cancer Res. 75 (22), 4790-4802 (2015).</li><li>Khurshed, M., Molenaar, R. J., Lenting, K., Leenders, W. P., van Noorden, C. J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+silico+gene+expression+analysis+reveals+glycolysis+and+acetate+anaplerosis+in+IDH1+wild-type+glioma+and+lactate+and+glutamate+anaplerosis+in+IDH1-mutated+glioma.">In silico gene expression analysis reveals glycolysis and acetate anaplerosis in IDH1 wild-type glioma and lactate and glutamate anaplerosis in IDH1-mutated glioma.</a> Oncotarget. 8 (30), 49165-49177 (2017).</li><li>Alberts, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular biology of the cell. , (2015).</li><li>Berg, J. M., Tymoczko, J. L., Gatto, G. J., Stryer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biochemistry. , (2015).</li><li>Webb, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enzyme nomenclature 1992 : recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes. , (1992).</li><li>Botman, D., Tigchelaar, W., Van Noorden, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Phosphate-activated+Glutaminase+Activity+and+Its+Kinetics+in+Mouse+Tissues+using+Metabolic+Mapping+(Quantitative+Enzyme+Histochemistry).">Determination of Phosphate-activated Glutaminase Activity and Its Kinetics in Mouse Tissues using Metabolic Mapping (Quantitative Enzyme Histochemistry).</a> J Histochem Cytochem. 62 (11), 813-826 (2014).</li><li>van Lith, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+as+chemotactic+fuel+for+diffuse+glioma+cells:+are+they+glutamate+suckers?.">Glutamate as chemotactic fuel for diffuse glioma cells: are they glutamate suckers?.</a> Biochim Biophys Acta. 1846 (1), 66-74 (2014).</li></ol>

作者没有什么可透露的。

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

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

<table border="0" cellpadding="0" cellspacing="0" width="972">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Adenosine 5&#8242;-diphosphate sodium salt</td>
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			<td height="21" style="height:21px;width:343px;">D-Glucose 6-phosphate sodium salt</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7879</td>
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			<td height="25" style="height:25px;width:343px;">Disodium hydrogen phosphate (NA2HPO4)</td>
			<td style="width:343px;">Merck Millipore</td>
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			<td height="21" style="height:21px;width:343px;">di-Sodium succinate</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:119px;">W327700</td>
			<td></td>
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			<td height="21" style="height:21px;width:343px;">DL-Isocitric acid trisodium salt hydrate</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:119px;">I1252&#160;</td>
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			<td height="21" style="height:21px;width:343px;">D-Malic acid</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">2300</td>
			<td></td>
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			<td height="21" style="height:21px;width:343px;">Glycerol Gelatin</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:119px;">GG1</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:343px;">L-Glutamic acid</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1251</td>
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			<td height="21" style="height:21px;width:343px;">Lithium 6-phospho-D-galactonate</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">55962</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Magnesium chloride</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:343px;">methoxy-Phenazine methosulfate</td>
			<td style="width:343px;">Serva</td>
			<td style="width:119px;">28966.01</td>
			<td></td>
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			<td height="21" style="height:21px;width:343px;">Nicotinamide adenine dinucleotide</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:119px;">N0505&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Nicotinamide adenine dinucleotide phosphate</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:119px;">NADP-RO</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Nitrotetrazolium Blue chloride</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:119px;">N6876</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Phenazine methosulfate</td>
			<td style="width:343px;">Serva</td>
			<td style="width:119px;">32030.01</td>
			<td></td>
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			<td height="21" style="height:21px;width:343px;">Polyvinyl alcohol, fully hydrolyzed</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:119px;">P1763</td>
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			<td height="25" style="height:25px;width:343px;">Potassium dihydrogen phosphate (KH2PO4)</td>
			<td style="width:343px;">Merck Millipore</td>
			<td align="right" style="width:119px;">104873</td>
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			<td height="21" style="height:21px;width:343px;">Sodium azide</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2002</td>
			<td></td>
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			<td height="21" style="height:21px;width:343px;">Sodium L-lactate</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:119px;">L7022</td>
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			<td height="21" style="height:21px;width:343px;">Bandpass filter</td>
			<td style="width:343px;">Edmund optics</td>
			<td style="width:119px;"></td>
			<td>https://www.edmundoptics.de/optics/optical-filters/bandpass-filters/Hard-Coated-OD-4-10nm-Bandpass-Filters/</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Grey-step wedge</td>
			<td style="width:343px;">Stouffer Industries</td>
			<td style="width:119px;"></td>
			<td>http://www.stouffer.net/TransPage.htm</td>
		</tr>
	</tbody>
</table>

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

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

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

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

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

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

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

代谢图谱: 定量酶化学和组织化学法测定脱氢酶在细胞和器官中的活性

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

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

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

科研

免疫与感染

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

在无菌无鼠标进定植评估肝代谢的变化 1 H核磁共振光谱

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

方法来抑制细菌Pyomelanin生产和确定敏感性氧化应激的影响相应增加

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

原位MHC-聚丙烯染色和定量分析确定抗原特异性 CD8 T 细胞在组织中的位置、丰度和表型

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

代谢图谱: 定量酶化学和组织化学法测定脱氢酶在细胞和器官中的活性

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星状细胞的七个步骤

在细胞命运

在无菌无鼠标进定植评估肝代谢的变化¹ H核磁共振光谱

量

方法来抑制细菌Pyomelanin生产和确定敏感性氧化应激的影响相应增加

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

定量微中和试验的优化

原位MHC-聚丙烯染色和定量分析确定抗原特异性 CD8 T 细胞在组织中的位置、丰度和表型

人类的过表达和纯化

一种新型大鼠自然杀伤细胞无饲养系统的研究体外