Name: analysis of the lipid composition of mycobacteria by thin layer chromatography
Uploaded: 2021-04-16T13:00:00
Description: Watch this Scientific Journal Video about Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography at JoVE.com

这项研究由西班牙科学、创新和大学部（RTI2018-098777-B-I00）、联邦基金和加泰罗尼亚总务局（2017SGR-229）资助。桑德拉·瓜拉尔-加里多是加泰罗尼亚总务部的博士学位合同（FI）的接受者。

1. 从分枝杆菌中提取总非同价相关脂质（图1）
<ol><li>从固体介质中划出 0.2 克分枝杆菌，并加入带多氟乙烯 （PTFE） 衬里螺丝帽的玻璃管。添加由 5 mL 氯仿和 10 mL 甲醇（氯仿：甲醇，1：2）组成的溶液。 注：使用有机溶剂时，只应使用玻璃接收器。不允许使用塑料容器。此外，需要为瓶子的PTFE衬里螺丝帽。 警告：氯仿是一种潜在的有毒和极其危险的物质。它必须使?...

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提出了一个简单的协议，被认为是从分不育细胞壁中提取非高血脂化合物的黄金标准方法。显示从四种不同分枝杆菌提取的脂质中，一维和二维 TLC 的进一步可视化。
氯仿和甲醇连续两次结合混合物来恢复支核细胞的脂质含量，是使用最广泛的溶剂混合物23、24、25、26、27、28、29。</su...

Mycobacterium 属包括致病性物种和非致病性物种，其特点是具有由其特有的脂质形成的高度疏水性和不透水性细胞壁。具体来说，分枝杆菌细胞壁含有霉菌酸， 这是α-烷基和β-羟基脂肪酸，其中α分支是恒定的所有肌酸（长度除外）和β链，称为中分子链，是一个长的碱性链，可能包含不同的功能化学组描述与文献（α，α'-，甲氧基， [-，环氧树脂，卡盒-和+-1-甲氧-肌酸），因此产生七种类型的肌酸（I-VII）1.此外，其他具有毫无疑问重要性的脂质也存在于分枝杆菌物种的细胞壁中。致病物种，如 Mycobacterium tuberculosis, 结核病的致病剂2 产生特定的脂基毒性因子，如邻苯二甲酸二甲酸酯 （PDIM）、酚类糖脂 （PGL）、二、三和五氯乙酰丙烯酸酯 （DAT、TAT 和 PAT） 或硫化物等3.它们在分不育表面的存在与改变宿主免疫反应的能力有关，因此，宿主内部的分不分母的进化和持久性4.例如，三乙基甘油 （TAG） 的存在与血统 2- 北京子谱的超病毒表型有关 M. tuberculosis, 可能是因为它能够减弱宿主的免疫反应5,6.其他相关的脂质是结块状和非结管分枝杆菌中的脂糖（LOSs）。在 Mycobacterium marinum，LOS在其细胞壁的存在与滑动运动和形成生物膜的能力有关，并干扰巨噬细胞模式识别受体的识别，影响宿主噬细胞吸收和消灭细菌7,8.此外，一些脂质的缺失或存在允许同一物种的成员在与宿主细胞相互作用时，被分为具有毒性或衰减型的不同体型。例如，在粗糙的太平间类型中缺少糖镜（GPL） Mycobacterium abscessus 已与诱导噬细胞内酸化的能力有关，因此细胞凋亡9，不像光滑的太平型，拥有GPL在他们的表面。此外，分机细胞壁的脂质含量与改变宿主免疫反应的能力有关。这在使用一些分枝杆菌触发针对不同病理的保护性免疫特征的背景下是相关的10,11,12,13. 例如，已经证明这一点 Mycolicibacterium vaccae，一种作为结核病免疫治疗疫苗进行第三阶段临床试验的沙氏杆菌，显示两种殖民的死型。虽然光滑的表型，其中含有聚酯在其表面，触发Th2反应，粗糙的表型没有聚酯可以诱导Th1配置文件时，它与宿主免疫细胞相互作用14.分枝杆菌细胞中存在的脂质的种类不仅取决于分枝杆菌物种，还取决于分枝杆菌培养的条件：孵化时间15,16 或文化媒介的构成17,18.事实上，培养媒介成分的变化影响着抗肿瘤和免疫刺激活性的 M. bovis BCG 和 Mycolicibacterium brumae in vitro17.此外，保护性免疫剖面触发 M. bovis BCG 反对 M. tuberculosis 小鼠模型的挑战也取决于文化媒介 M. bovis BCG 增长17.然后，这些可能与每个培养条件下的分枝杆菌的脂质组成有关。由于所有这些原因，对分枝杆菌脂质含量的研究是相关的。提出了提取和分析支点细胞壁脂质成分的视觉程序。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic Acid</td>
			<td>Merck</td>
			<td>100063</td>
			<td>CAUTION. Anhydrous for analysis EMSURE&#174; ACS,ISO,Reag. Ph Eur</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Carlo Erba</td>
			<td>400971N</td>
			<td>CAUTION. ACETONE RPE-ACS-ISO FOR ANALYS ml 1000</td>
		</tr>
		<tr>
			<td>Anthrone</td>
			<td>Merck</td>
			<td>8014610010</td>
			<td>Anthrone for synthesis.</td>
		</tr>
		<tr>
			<td>Benzene</td>
			<td>Carlo Erba</td>
			<td>426113</td>
			<td>CAUTION. Benzene RPE - For analysis - ACS 2.5 l</td>
		</tr>
		<tr>
			<td>Capillary glass tube</td>
			<td>Merck</td>
			<td>BR708709</td>
			<td>BRAND&#174;&#160;disposable BLAUBRAND&#174;&#160;micropipettes, intraMark</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Carlo Erba</td>
			<td>412653</td>
			<td>CAUTION. Chloroform RS - For HPLC - Isocratic grade - Stabilized with ethanol 2.5 L</td>
		</tr>
		<tr>
			<td>Dry block heater</td>
			<td>J.P. Selecta</td>
			<td>7471200</td>
			<td></td>
		</tr>
		<tr>
			<td>Dicloromethane</td>
			<td>Carlo Erba</td>
			<td>412622</td>
			<td>CAUTION. Dichloromethane RS - For HPLC - Isocratic grade - Stabilized with amylene 2.5 L</td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Carlo Erba</td>
			<td>412672</td>
			<td>CAUTION. Diethyl ether RS - For HPLC - Isocratic grade - Not stabilized 2.5 L</td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Panreac</td>
			<td>1313181211</td>
			<td>CAUTION. Ethyl acetate (Reag. USP, Ph. Eur.) for analysis, ACS, ISO</td>
		</tr>
		<tr>
			<td>Ethyl Alcohol Absolute</td>
			<td>Carlo Erba</td>
			<td>4146072</td>
			<td>CAUTION. Ethanol absolute anhydrous RPE - For analysis - ACS - Reag. Ph.Eur. - Reag. USP 1 L</td>
		</tr>
		<tr>
			<td>Glass funnel</td>
			<td>VidraFOC</td>
			<td>DURA.2133148 1217/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tube</td>
			<td>VidraFOC</td>
			<td>VFOC.45066A-16125</td>
			<td>Glass tube with PTFE recovered cap</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carlo Erba</td>
			<td>412722</td>
			<td>CAUTION. Methanol RS - For HPLC - GOLD - Ultragradient grade 2.5 L</td>
		</tr>
		<tr>
			<td>Molybdatophosphoric acid hydrate</td>
			<td>Merck</td>
			<td>51429-74-4</td>
			<td>CAUTION.</td>
		</tr>
		<tr>
			<td>Molybdenum Blue Spray Reagent, 1.3%</td>
			<td>Sigma</td>
			<td>M1942-100ML</td>
			<td>CAUTION.</td>
		</tr>
		<tr>
			<td>n-hexane</td>
			<td>Carlo Erba</td>
			<td>446903</td>
			<td>CAUTION. n-Hexane 99% RS - ATRASOL - For traces analysis 2.5 L</td>
		</tr>
		<tr>
			<td>n-nitroso-n-methylurea</td>
			<td>Sigma</td>
			<td>N4766</td>
			<td>CAUTION</td>
		</tr>
		<tr>
			<td>Orbital shaking platform</td>
			<td>DDBiolab</td>
			<td>995018</td>
			<td>NB-205L benchtop shaking incubator</td>
		</tr>
		<tr>
			<td>Petroleum ether (60-80&#186;C)</td>
			<td>Carlo Erba</td>
			<td>427003</td>
			<td>CAUTION. Petroleum ether 60 - 80&#176;C RPE - For analysis 2.5 L</td>
		</tr>
		<tr>
			<td>Sprayer</td>
			<td>VidraFOC</td>
			<td>712/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulphate anhydrous</td>
			<td>Merck</td>
			<td>238597</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid 95-97%</td>
			<td>Merck</td>
			<td>1007311000</td>
			<td>CAUTION. Sulfuric acid 95-97%</td>
		</tr>
		<tr>
			<td>TLC chamber</td>
			<td>Merck</td>
			<td>Z204226-1EA</td>
			<td>Rectangular TLC developing tanks, complete L &#215; H &#215; W 22 cm &#215; 22 cm &#215; 10 cm</td>
		</tr>
		<tr>
			<td>TLC plate</td>
			<td>Merck</td>
			<td>1057210001</td>
			<td>TLC SilicaGel 60- 20x20 cm x 25 u</td>
		</tr>
		<tr>
			<td>TLC Plate Heater</td>
			<td>CAMAG</td>
			<td>223306</td>
			<td>CAMAG TLC Plat Heater III</td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Carlo Erba</td>
			<td>488551</td>
			<td>CAUTION. Toluene RPE - For analysis - ISO - ACS - Reag.Ph.Eur. - Reag.USP 1 L</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>10132562</td>
			<td>IKA&#160;Agitador IKA v&#243;rtex 3</td>
		</tr>
		<tr>
			<td>1-naphthol</td>
			<td>Sigma-Aldrich</td>
			<td>102269427</td>
			<td>CAUTION.</td>
		</tr>
	</tbody>
</table>

analysis of the lipid composition of mycobacteria by thin layer chromatography

分枝杆菌物种在生长速度、色素递迫存在、固体介质上显示的群落形态以及其他表型特征方面可能各不相同。然而，它们都有一个共同的分枝杆菌最相关的特征：它独特的高度疏水细胞壁。分枝杆菌物种含有一种膜-共价连结复合物，包括阿拉比诺甘丹、多肽和长链的支肠酸，其类型不同于分枝杆菌物种。此外， 肌菌还可以在其细胞表面产生位于非共价链接的脂质，如邻苯二甲酸二酯（PDIM）、酚甘油脂（PGL）、甘油脂（GPL）、乙酰三聚醇（AT）或磷脂二恶英（PIM）等。其中一些被认为是致病性分枝杆菌的毒性因素，或宿主-分枝杆菌相互作用中的关键抗原脂。由于这些原因，对支点血脂的研究有着极大的兴趣，因为这种脂质在几个领域得到应用，从了解它们在分枝杆菌感染致病性中的作用，到可能暗示它们是治疗传染病和其他疾病（如癌症）的免疫调节剂。在这里，提出了一个简单的方法来提取和分析总脂质含量和用有机溶剂混合物生长在固体介质中的分枝杆菌细胞的肌酸成分。获得脂质提取物后，将执行薄层色谱 （TLC） 来监测提取的化合物。该示例实验采用四种不同的分枝杆菌进行：环境快速增长的肌 杆菌布鲁马和支原体杆菌， 生长缓慢的 肌杆菌博维斯 杆菌Calmette-Guérin（BCG）和机会型病原体快速生长的分 枝杆菌脓肿， 表明本协议中显示的方法可用于广泛的分枝杆菌。

为了显示不同分枝杆菌物种中的各种脂质 ，M. bovis BCG 被选中，因为它是粗糙和生长缓慢的分枝杆菌。在手术中增加了粗糙和快速增长的 M.偶然 和 M.布鲁马 ，最后，也包括 了M.脓肿 的光滑的死型。这四个物种使我们能够可视化广泛的分枝杆菌衍生脂质，如乙酰链球菌（AT）、GPL、PDIM、PGL、PIM、TDM和TMM。此外，所有四个物种都有不同的肌酸模式。
在...

Watch this Scientific Journal Video about Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography at JoVE.com

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

提出一个协议，以提取各种分枝杆菌细胞壁的总脂质含量。此外，还显示了不同类型的肌酸的萃取和分析协议。还提供了一个薄层色谱协议来监测这些分形化合物。

薄层色谱分析分枝杆菌的脂质成分

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...

分枝杆菌脂质是属的重要特征，在宿主分枝杆菌相互作用中至关重要。速度和多功能性是此过程的主要优点。该方法可用于了解脂质的作用和分枝杆菌的致病性及其免疫刺激作用。对于非共价连结脂质提取，在层压流罩下带有 PTFE 衬里螺丝盖的玻璃管中加入 15 毫升一至两个氯仿到甲醇溶液中。然后，从固体介质中划出200毫克的分枝杆菌，并将分枝杆菌添加到玻璃管中。用不断的搅拌在一夜之间孵育管子。第二天，通过玻璃漏斗将有机溶剂过滤成玻璃管，该漏斗内衬过滤纸。使用氮气通量后，蒸发管中的液相，用氮气填充管子，将管子储存在摄氏四度。然后，在细胞碎屑中加入 15 毫升 2 到 1 氯仿的甲醇溶液，并通过不断搅拌在一夜之间孵育管子。第二天早上，让混合物休息一小时，然后使用巴斯德移液器将有机溶剂转移到过滤纸衬里玻璃漏斗中，放入同一个玻璃收集管中。然后，蒸发液相，如所示，然后用氮气重新填充管中四摄氏度的储存。对于肌酸提取，在带PTFE衬里螺丝盖的密封玻璃管中加入2至5毫升的搅拌溶液和200毫克的分枝杆菌生物量，并通过涡流混合内含物。在80摄氏度的干燥浴池中将混合物孵化过夜。第二天，当管子冷却室温时，加入两毫升N六烷，并通过漩涡混合30秒。让管子沉淀，直到出现两个清晰的相位，并将上 N 六烷相转移到新管中。将另外两毫升N六烷与管内含物混合，并再次收集上层。然后，通过氮流蒸发管内的成分，并将覆盖在氮气中的样品储存在摄氏四度。如所示。要通过 TLC 将样品用一块滤纸覆盖 TLC 腔室的一面墙，请在腔室角添加凡士林来密封腔室，将溶剂混合物涂在过滤纸上，并将溶剂的剩余体积添加到 TLC 室的底部。关闭 TLC 室至少 20 分钟以饱和。同时，溶解200至1000微升氯仿玻璃管中的脂质，并使用毛细管玻璃管将10微升脂质氯仿悬浮剂直接涂抹在TLC板上。允许样品干燥五分钟后，将板插入饱和 TLC 室，并允许移动相流穿过 TLC。当溶剂到达离板的上端一厘米时，将板置于层压通量下，直到二氧化硅完全干燥。然后，在乙醇中用 15 到 20 毫升 10% 摩尔数据磷酸水合物喷洒干板，直到板为亮黄色，并在 120 摄氏度下加热板两到五分钟。在这种具有代表性的分析中，利用两种不同的脱液系统和二维TLC，通过一维TLC对从各种分枝杆菌中提取的支核酸进行了分析。还进行了两种甲基化程序，以确认五型肌酸的存在。在这两个分化系统中，肌酸大约位于板的中间，尽管，与五型肌酸对应的点，只有在皂化后才被观察到。二维TLC还允许识别单个肌酸类型。例如，支核酸只表示一型肌酸，肌杆菌BCG表示一型和四型，分子细菌表示一型和五型，分不分母体表示一型和二型肌酸。从各种分枝杆菌物种的总脂质提取物被分析在其极性和大小的功能。它揭示了大多数A极地脂质，如PDIM，存在于麦芽草，但不是在分温杆菌，分核酸菌布鲁马和M abcesus的光滑的太平缓型。酚酸甘油脂只存在于BCG的分细菌中，而糖肽只在分不育的样品中观察到。与霉菌酸类似，乙酰甘油和 PDIM、PIM 也可以通过一维 TLC 或 2D TLC 分析轻松可视化。使用两种类型的污渍在一维 TLC 中显示 PIM。在尝试此协议时，要记住的最重要的事情是在整个过程中不要使用任何塑料仪器。

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A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

科研

免疫与感染

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

上皮损伤制作分析<em&gt;阿米巴</em&gt;感染

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

木炭琼脂刃天青分析的可视化分枝杆菌的半定量，中等吞吐量枚举

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

微生物菌落生物膜的石蜡嵌入和薄切片显微分析

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing

使用自动图像处理分析裂变和萌芽酵母中的脂滴含量

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are ...

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

铁限分枝杆菌产生的细胞外囊泡的分离和特征

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...