Name: lipid index determination by liquid fluorescence recovery in the fungal pathogen ustilago maydis
Uploaded: 2018-04-03T15:00:00
Description: Watch this Scientific Journal Video about Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis at JoVE.com

这项工作得到了 Politécnico 国立 Secretaria de Investigación y Posgrado (IPN-SIP-20170864)、方案 de 支持农场 Proyectos-Investigación e Innovación Tecnológica (PAPIIT IN222117)-国立大学国立墨西哥的赠款的支持 (UNAM) 和委员会 Ciencia y Tecnología (CONACyT 254904-JPP 和 256520-GGS)。Fundação de 宪法保护 Pesquisa 做里约热内卢 (FAPERJ Cientistas 做 Nosso: E 26/103.353/2011)。我们感谢 QFB。奥斯卡豪尔赫·伊万·莫拉·戈多伊 Luqueño Bocardo 为示意图概述。我们感谢米格尔博士 Tapia 罗德里格斯在 LDs 的共焦显微术中的宝贵帮助。我们要感谢布鲁诺 Bozaquel Morais 博士在开发酵母技术方面的开创性工作, 我们已经适应了U. 玉米。

1. 缓冲和解决方案的准备工作
<ol><li>制备 1 L 磷酸缓冲盐水 (PBS, pH 值 7), 溶解8克氯化钠, 0.2 克氯化钾, 1.44 克 Na2HPO4, 0.24 g 的馏分2PO4在800毫升的水。用 HCl 将 pH 值调整为 7.0, 然后在1升的最终容积中加水. PBS 可作为10x 库存解决方案, 并存储在室温下。</li>
	<li>要准备10毫升的固定溶液, 添加1毫升37% 甲醛到9毫升的 PBS, 达到最后浓度的3.7%。请在使用前准备此解决方案。...

<ol>
	<li>Guo, Y., Cordes, K. R., Farese, R. V., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+at+a+glance.">Lipid droplets at a glance.</a> Journal of Cell Science. 122 (6), 749-752 (2009).</li><li>Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+roles+for+lipid+droplets.">Expanding roles for lipid droplets.</a> Current Biology. 25 (11), R470-R481 (2015).</li><li>Sestric, R., Munch, G., Cicek, N., Sparling, R., Levin, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+neutral+lipid+synthesis+by+Yarrowia+lipolytica+on+various+carbon+substrates+under+nutrient-sufficient+and+nutrient-limited+conditions.">Growth and neutral lipid synthesis by Yarrowia lipolytica on various carbon substrates under nutrient-sufficient and nutrient-limited conditions.</a> Bioresource Technology. 164, 41-46 (2014).</li><li>Raimondi, S., 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=Getting+lipids+from+glycerol:+new+perspectives+on+biotechnological+exploitation+of+Candida+freyschussii.">Getting lipids from glycerol: new perspectives on biotechnological exploitation of Candida freyschussii.</a> Microbial Cell Factories. 13, 83 (2014).</li><li>Cescut, J., Fillaudeau, L., Molina-Jouve, C., Uribelarrea, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+accumulation+in+Rhodotorula+glutinis+induced+by+nitrogen+limitation.">Carbon accumulation in Rhodotorula glutinis induced by nitrogen limitation.</a> Biotechnology for Biofuels. 7, 164 (2014).</li><li>Galafassi, S., 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=Lipid+production+for+second+generation+biodiesel+by+the+oleaginous+yeast+Rhodotorula+graminis.">Lipid production for second generation biodiesel by the oleaginous yeast Rhodotorula graminis.</a> Bioresource Technology. 111, 398-403 (2012).</li><li>Kot, A. M., Blazejak, S., Kurcz, A., Gientka, I., Kieliszek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodotorula+glutinis-potential+source+of+lipids,+carotenoids,+and+enzymes+for+use+in+industries.">Rhodotorula glutinis-potential source of lipids, carotenoids, and enzymes for use in industries.</a> Applied Microbiology and Biotechnology. 100 (14), 6103-6117 (2016).</li><li>Rakicka, M., Lazar, Z., Dulermo, T., Fickers, P., Nicaud, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+production+by+the+oleaginous+yeast+Yarrowia+lipolytica+using+industrial+by-products+under+different+culture+conditions.">Lipid production by the oleaginous yeast Yarrowia lipolytica using industrial by-products under different culture conditions.</a> Biotechnology for Biofuels. 8, (2015).</li><li>Zhu, Z. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+Lipid+Droplet+Proteome+of+the+Oleaginous+Yeast+Rhodosporidium+toruloides.">Dynamics of the Lipid Droplet Proteome of the Oleaginous Yeast Rhodosporidium toruloides.</a> Eukaryotic Cell. 14 (3), 252-264 (2015).</li><li>Kolouchova, I., Mat'atkova, O., Sigler, K., Masak, J., Rezanka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Palmitoleic+and+Linoleic+Acid+in+Oleaginous+and+Nonoleaginous+Yeast+Biomass.">Production of Palmitoleic and Linoleic Acid in Oleaginous and Nonoleaginous Yeast Biomass.</a> International Journal of Analytical Chemistry. , (2016).</li><li>Radulovic, M., 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=The+emergence+of+lipid+droplets+in+yeast:+current+status+and+experimental+approaches.">The emergence of lipid droplets in yeast: current status and experimental approaches.</a> Current Genetics. 59 (4), 231-242 (2013).</li><li>Takahashi, Y., 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=Perilipin-Mediated+Lipid+Droplet+Formation+in+Adipocytes+Promotes+Sterol+Regulatory+Element-Binding+Protein-1+Processing+and+Triacylglyceride+Accumulation.">Perilipin-Mediated Lipid Droplet Formation in Adipocytes Promotes Sterol Regulatory Element-Binding Protein-1 Processing and Triacylglyceride Accumulation.</a> Plos One. 8 (5), e64605 (2013).</li><li>Thiam, A. R., Farese Jr, R. V., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biophysics+and+cell+biology+of+lipid+droplets.">The biophysics and cell biology of lipid droplets.</a> Nature Reviews Molecular Cell Biology. 14 (12), 775-786 (2013).</li><li>Penno, A., Hackenbroich, G., Thiele, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipids+and+lipid+droplets.">Phospholipids and lipid droplets.</a> Biochimica et Biophysica Acta. 1831 (3), 589-594 (2013).</li><li>Ageitos, J. M., Vallejo, J. A., Veiga-Crespo, P., Villa, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oily+yeasts+as+oleaginous+cell+factories.">Oily yeasts as oleaginous cell factories.</a> Applied Microbiology and Biotechnology. 90 (4), 1219-1227 (2011).</li><li>Athenstaedt, K., Daum, 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+life+cycle+of+neutral+lipids:+synthesis,+storage+and+degradation.">The life cycle of neutral lipids: synthesis, storage and degradation.</a> Cellular and Molecular Life Sciences. 63 (12), 1355-1369 (2006).</li><li>Marshall, L. L., Stimpson, S. E., Hyland, R., Coorssen, J. R., Myers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+lipid+droplet+accumulation+associated+with+a+peripheral+sensory+neuropathy.">Increased lipid droplet accumulation associated with a peripheral sensory neuropathy.</a> Journal of Biological Chemistry. 7 (2), 67-76 (2014).</li><li>Filipe, A., McLauchlan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+and+lipid+droplets:+finding+a+niche.">Hepatitis C virus and lipid droplets: finding a niche.</a> Trends in Molecular Medicine. 21 (1), 34-42 (2015).</li><li>Greenberg, A. S., 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=The+role+of+lipid+droplets+in+metabolic+disease+in+rodents+and+humans.">The role of lipid droplets in metabolic disease in rodents and humans.</a> Journal of Clinical Investigation. 121 (6), 2102-2110 (2011).</li><li>Aguilar, L. R., 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=Lipid+droplets+accumulation+and+other+biochemical+changes+induced+in+the+fungal+pathogen+Ustilago+maydis+under+nitrogen-starvation.">Lipid droplets accumulation and other biochemical changes induced in the fungal pathogen Ustilago maydis under nitrogen-starvation.</a> Archives of Microbiology. , (2017).</li><li>Bozaquel-Morais, B. L., Madeira, J. B., Maya-Monteiro, C. M., Masuda, C. A., Montero-Lomeli, 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+new+fluorescence-based+method+identifies+protein+phosphatases+regulating+lipid+droplet+metabolism.">A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.</a> PLoS One. 5 (10), e13692 (2010).</li><li>Madeira, J. B., 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=TORC1+Inhibition+Induces+Lipid+Droplet+Replenishment+in+Yeast.">TORC1 Inhibition Induces Lipid Droplet Replenishment in Yeast.</a> Molecular and Cellular Biology. 35 (4), 737-746 (2015).</li><li>Maya-Monteiro, C. M., 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=Leptin+induces+macrophage+lipid+body+formation+by+a+phosphatidylinositol+3-kinase-+and+mammalian+target+of+rapamycin-dependent+mechanism.">Leptin induces macrophage lipid body formation by a phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent mechanism.</a> Journal of Biological Chemistry. 283 (4), 2203-2210 (2008).</li><li>Listenberger, L. L., Brown, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Cell Biology. , (2001).</li><li>Jump, D. B., Torres-Gonzalez, M., Olson, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soraphen+A,+an+inhibitor+of+acetyl+CoA+carboxylase+activity,+interferes+with+fatty+acid+elongation.">Soraphen A, an inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation.</a> Biochemical Pharmacology. 81 (5), 649-660 (2011).</li><li>Hobro, A. J., Smith, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+fixation+methods:+Spatial+and+compositional+cellular+changes+observed+by+Raman+imaging.">An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging.</a> Vibrational Spectroscopy. 91, 31-45 (2017).</li><li>Chen, Y. T., Wan, L., Zhang, D. P., Bian, Y. Z., Jiang, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+spectroscopic+property+of+Bodipy+derivates+through+tuning+the+molecular+configuration.">Modulation of the spectroscopic property of Bodipy derivates through tuning the molecular configuration.</a> Photochemical &amp; Photobiological Sciences. 10 (6), 1030-1038 (2011).</li><li>Pagac, M., 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=SEIPIN+Regulates+Lipid+Droplet+Expansion+and+Adipocyte+Development+by+Modulating+the+Activity+of+Glycerol-3-phosphate+Acyltransferase.">SEIPIN Regulates Lipid Droplet Expansion and Adipocyte Development by Modulating the Activity of Glycerol-3-phosphate Acyltransferase.</a> Cell Reports. 17 (6), 1546-1559 (2016).</li><li>Qiu, B., Simon, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BODIPY+493/503+Staining+of+Neutral+Lipid+Droplets+for+Microscopy+and+Quantification+by+Flow+Cytometry.">BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry.</a> Bio-protocol. 6 (17), (2016).</li><li>Rumin, 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=The+use+of+fluorescent+Nile+red+and+BODIPY+for+lipid+measurement+in+microalgae.">The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae.</a> Biotechnology for Biofuels. 8, 42 (2015).</li><li>Holliday, R., King, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ustilago+maydis.">Ustilago maydis.</a> Handbook of genetics. , 575-595 (1974).</li></ol>

LFR 是首次应用于研究酵母中脂质代谢的新方法, 后来在U. 玉米20,21中成功实现。虽然 ld 指数并没有给出细胞中累积的标记的绝对值, 但它为细胞的脂质含量提供了一个快速的概念, 当比较两个或多个实验条件的 LD 指数时, 对数据的解释是直截了当的。BODIPY 493/503 是高度特异的中性脂, 主要成分的 LDs, 它有一个狭窄的发射频谱, 以促进同时检测...

脂滴 (LDs) 是无处不在的细胞内脂肪体组成的核心中性脂, 主要是标签和甾醇酯 (SE)。核心周围有一层磷脂, 与蛋白质如 perilipin 和酶参与合成中性脂, 如甘油酰基转移酶, 乙酰基 coa 酶和酰 coa 综合体,1。由于其动态行为, 磷脂单层还含有水解标记和 SE2的三酰甘油脂肪酶。根据细胞类型和有机体, 储存中性脂可以用来产生能量, 或合成磷脂和信号分子。在酵母和其他真菌中, LDs 的含量随培养基中氮/碳比值的变化而变化, 表明氮的可用性降低可能是提高中性脂产量的关键3,4 ,5。在酵母中生产大量的标签, 在生物技术中作为生物燃料的来源和食品工业中有潜在的用途。一些储存的脂质含有高比例的多不饱和脂肪酸, 如欧米茄3和欧米茄 6, 营养和膳食重要性6,7,8,9,10,11. 包括人类细胞在内的哺乳动物细胞的 LDs 也含有标记和胆固醇酯。磷脂单层与酵母 LDs 中描述的蛋白质的哺乳动物同源性相互作用, 另外一种蛋白质, perilipin, 在酵母12中不存在。磷脂单层及其相关蛋白的一个建议作用是稳定 lds 结构, 并允许 lds 与细胞器的相互作用, 如线粒体、内质网、过氧化物酶体和液泡, 主要用于脂交换13,14。有趣的是, 在人类中, LDs 似乎参与了2型糖尿病, 动脉粥样硬化, 脂肪性肝炎和冠心病的病理, 其中有增加的数字15,16,17.某些类型的病毒使用 LDs 作为平台来组装病毒2,18,19。
由于 lds 在人类病理中的影响及其潜在的生物技术应用, 对 lds 形成的精确实验测定是一项重要的任务。本文描述了一个可靠的检测方法的基础上恢复荧光 (LFR) 的 BODIPY 493/503 (4, 4-difluoro-1, 3, 5, 7, 8-pentamethyl-4-波拉-3 a, 4 a-diaza s indacene), 以获得相对价值的中性脂在细胞中的含量。通过这种分析, 可以跟踪真菌中中性脂类积累的动态, 如U. 玉米和酿酒酵母, 还有哺乳动物细胞, 不需要脂质提取20.该方法首次在酿酒酵母中应用, 以确定参与调节脂质代谢的蛋白磷酸和激酶。这是可能的, 不用油脂提取或蛋白质纯化21,22。LFR 也被用来建立腹腔巨噬细胞的 LDs 形成的动力学23。BODIPY 493/503 的使用比其他中性脂质染料有一定的优势, 如尼罗河红色。BODIPY 493/503 是高度特异的中性脂质和有一个狭窄的放射频谱, 促进同时检测的信号, 如红色荧光蛋白或图面-跟踪, 当样品分析的共焦显微镜。不幸的是, BODIPY 493/503 对漂白很敏感, 但在暴露于光24中使用 antiquenching 试剂可以避免此过程。
在酵母细胞中进行 LFR 测定, 在理想的营养条件下培养, 整除数在不同时间被提取。其次, 细胞是固定的甲醛, 这保持了 LDs 的完整性数月, 当细胞存储在4摄氏度。其他固定技术应避免, 特别是使用甲醇或冷丙酮, 因为它们导致 LDs 在细胞内的退化24。为了测量 LD 指数, 甲醛固定细胞悬浮在水中, 以获得明确的浓度。然后, 它们被添加到包含荧光 BODIPY 493/503 的解决方案中, 当荧光进入细胞并与 LDs 关联时, 它的荧光就会恢复。伴随荧光测量 (485 nm/510 nm), 细胞的浓度通过测量 600 nm 的光学密度来量化。每一个样品被读四次通过增加随后5µL 整除数甲醛固定的细胞悬浮到同样井。荧光和吸光度的空白在细胞的加法之前获得。通过确定测量的线性度来评估荧光和吸光度数据的质量: 如果 r < 0.9, 数据被丢弃。r 值是重要的, 因为基本上荧光强度应该产生一个线性反应, 以增加细胞浓度。如果细胞浓度过高, 线性度就会丢失。LFR 法为高通量实验中选择所需的 LD 表型提供了一种快速、简便、经济的方法。在选择所需条件后, 可以通过共焦显微镜对单个细胞的 LD 含量进行研究, 使用与 BODIPY 染色的相同的甲醛固定细胞, 提供细胞中 LDs 的图像。用薄层色谱法进一步分析了它们的标记和硒含量。

<table>
	<tbody>
		<tr>
			<td>Spectrophotometer</td>
			<td>Varioskan Lux multimode microplate reade</td>
			<td>D5879</td>
			<td>Filter 493/503 or monochromator detector</td>
		</tr>
		<tr>
			<td>Plate costar</td>
			<td>Corning Inc.</td>
			<td>3615</td>
			<td>96 well black-wall/clear bottom</td>
		</tr>
		<tr>
			<td>Plate costar&#160;</td>
			<td>Corning Inc</td>
			<td>3598</td>
			<td>96 well cell culture plate costar</td>
		</tr>
		<tr>
			<td>BODIPY 493/503</td>
			<td>Invitrogen/ Thermofisher</td>
			<td>D3922</td>
			<td>4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene</td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma Aldrich</td>
			<td>252549</td>
			<td>ACS reagent, 37 wt % in H2O, contains 10-15% methanol to prevent polymerization</td>
		</tr>
		<tr>
			<td>Potassium iodide</td>
			<td>Sigma Aldrich</td>
			<td>60399</td>
			<td>BioUltra, &#8805; 99.5% (AT)</td>
		</tr>
		<tr>
			<td>FB2 Ustilago maydis</td>
			<td>ATCC</td>
			<td>201384</td>
			<td>Basidiomycete-Yeast</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>746398</td>
			<td>ACS reagent, inorganic salt</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>P0662</td>
			<td>ACS reagent</td>
		</tr>
		<tr>
			<td>Select Yeast Extract</td>
			<td>Sigma Aldrich</td>
			<td>Y100</td>
			<td>Mixture of amini acids, peptides, water- soluble, vitamins and carbohydrates for culture media</td>
		</tr>
		<tr>
			<td>N-Z case plus</td>
			<td>Sigma aldrich</td>
			<td>N4642</td>
			<td>Casein enzymatic hydrolyzate from bovine milk</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G-8270</td>
			<td>D (+) glucose</td>
		</tr>
		<tr>
			<td>Skaker flask</td>
			<td>Pirex</td>
			<td>CLS4450250-6EA</td>
			<td>Borosiicate glass</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>SEV M&#233;xico</td>
			<td>INO650V-7</td>
			<td>Orbital shaker</td>
		</tr>
		<tr>
			<td>Centrifuge table</td>
			<td>Eppendorf</td>
			<td>5415C</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Sigma Aldrich</td>
			<td>Z606340</td>
			<td>Eppendorf</td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>Axygen scientific</td>
			<td>T-200-Y</td>
			<td>Universal Pipet Tips with Bevelled End, 200 microliter, non sterile</td>
		</tr>
		<tr>
			<td>mLine pipette</td>
			<td>Biohit</td>
			<td>725130</td>
			<td>8 channels, volume range 5-100 uL</td>
		</tr>
	</tbody>
</table>

lipid index determination by liquid fluorescence recovery in the fungal pathogen ustilago maydis

本文介绍了应用 LD 指数法测定脂滴 (LDs) 中甘油三酯 (微板块) 积累的灵敏的检测方法。获得了不带脂质提取的 LD 指数。它允许测量在不同条件下的高吞吐量实验中的 LDs 含量, 如富含或氮气耗尽介质的生长。尽管该方法首次被描述为研究酿酒酵母中的脂滴代谢, 它成功地应用于担子黑穗病玉米。有趣的是, 由于 LDs 是真核细胞 phylogenetically 保存的细胞器, 这种方法可以应用于大量的不同的干细胞, 从酵母到哺乳动物细胞。LD 指数是基于 BODIPY 493/503 在淬火条件下的液体荧光恢复试验 (LFR), 通过添加与甲醛固定的细胞.碘钾用作荧光淬火。荧光与光密度斜率的比值为 LD 指数。斜坡是计算的直线, 当 BODIPY 荧光和光学密度在 600 nm (OD600) 绘制的样本添加。最佳数据质量是由相关系数等于或高于 0.9 (r ≥ 0.9) 反映出来的。可以同时读取多个示例, 因为它可以在微板块中实现。由于 BODIPY 493/503 是一种脂溶性荧光染料, 分成脂肪滴, 它可以用于许多类型的细胞积累 LDs。

LFR, 与 BODIPY 493/503 作为荧光探针是一个可靠和容易的方法来研究 LDs 在U. 玉米,的动态积累, 无论生长条件如何。当细胞在 YPD 培养基中培养时, LD 指数在指数阶段增加, 其次是固定相的下降 (图 2A)。与此相反, 在氮饥饿下生长的细胞中, LD 指数在指数和固定相位上都有稳步增加 (图 2B)。由于两种区域性都是以相同的光密度 (?...

Watch this Scientific Journal Video about Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis at JoVE.com

Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

在这里, 我们描述了一个获得脂滴指数 (LD 指数) 的协议, 以研究在高通量实验中培养的细胞中甘油三酯的动力学。LD 指数法是一种使用 BODIPY 493/503 的简便可靠的方法。这种检测不需要 dispendious 脂提取或显微分析。

真菌病原体液体荧光恢复法测定脂质指数黑穗病玉米

The article shows how to implement the LD index assay, which is a sensitive microplate assay to determine the accumulation of triacylglycerols (TAGs) in ...

Research

JoVE Journal

Biochemistry

Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Determination of the Glycogen Content in Cyanobacteria

测定蓝细菌中的糖原含量

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

酶活化致癌物和药物对共价 DNA 加合物的生成研究体外及其对32 P-postlabeling 的测定

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

MALDI 质谱法测定小鼠组织提取物蛋白酶特异性: 操作 PH 值导致特异性改变

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

通过质量光度法快速测定抗体抗原亲和力

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

肽聚糖的液相色谱质谱和生物信息学半定量分析

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

酶的结晶和结构确定：多功能微流体芯片中串行晶体学的基板复合物

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...