Name: single-cell analysis of bacillus subtilis biofilms using fluorescence microscopy and flow cytometry
Uploaded: 2012-02-15T12:00:00
Description: Watch this Scientific Journal Video about Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry at JoVE.com

这项工作是由青年研究者的研究计划，从维尔茨堡大学传染病研究中心（ZINF）。胡安·Ç加西亚贝坦库尔是从维尔茨堡大学生命科学院研究生院（GSLS）博士研究员。

1。标签B。枯草和生物膜形成实验<ol><li>通过PCR扩增感兴趣的基因的启动子区域。我们的P TAPA，负责生产的的TASA基质蛋白26基因启动子的克隆为例。克隆带够TAPA到pkm008向量（Rudner实验室，哈佛医学院，波士顿，美国）（图2）。 </li><li>线性质粒，通过消化酶（酶建议，XhoI位）。 </li><li> 在 B诱导自然能力枯草芽孢杆菌菌株按照协议，先前?...

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Microbiol. 74, 609-618 (2009).</li><li>Arima, K., Kakinuma, A., Tamura, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactin,+a+crystalline+peptidelipid+surfactant+produced+by+Bacillus+subtilis:+isolation,+characterization+and+its+inhibition+of+fibrin+clot+formation.">Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation.</a> Biochem. Biophys. Res. Commun. 31, 488-494 (1968).</li><li>Romero, D., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+accessory+protein+required+for+anchoring+and+assembly+of+amyloid+fibres+in+B.+subtilis+biofilms.">An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms.</a> Mol. 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事实上，细菌群落表明细胞亚群的表达一组特定的基因证据33,34微生物群落的复杂性。本议定书应帮助，以确定是否对任何感兴趣的基因的表达仅限于微生物群落，特别是一个专门的细胞亚群。这些亚群的可视化需要新技术的发展，因为传统的方法来监测基因表达微阵列基因表达分析率水平，整个微生物群落内的微生物群落的基因表达的波动一般失手。 荧光显微镜和?...

<table border="1"><tbody><tr><td> 技术 </td><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td rowspan="11"> MSgg组成</td><td>磷酸钾5MM </td><td>罗斯</td><td> 6878 </td></tr><tr><td>公开资讯观测站100MM </td><td> Sigma-Aldrich公司</td><td> M1254 </td></tr><tr><td>氯化镁2MM </td><td>罗斯</td><td> 2189.1 </td></tr><tr><td>钙氯化物70​​0μM </td><td>罗斯</td><td> A119.1 </td></tr><tr><td>三氯化铁50μM </td><td> Sigma-Aldrich公司</td><td> 157740 </td></tr><tr><td>锌氯1μM </td><td> applichem </td><td> A2076 </td></tr><tr><td>硫胺素2μM </td><td> Sigma-Aldrich公司</td><td> 74625 </td></tr><tr><td>甘油0.5％ </td><td>罗斯</td><td> 7533 </td></tr><tr><td>谷氨酸队友0.5％ </td><td> Sigma-Aldrich公司</td><td> 49621 </td></tr><tr><td>色氨酸为50μg/ml </td><td> Sigma-Aldrich公司</td><td> T0254 </td></tr><tr><td>苯丙氨酸为50μg/ml </td><td> Sigma-Aldrich公司</td><td> P2126 </td></tr><tr><td>细胞固定</td><td>多聚甲醛</td><td>罗斯</td><td> 0335 </td></tr><tr><td></td><td> 设备名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>超声</td><td>细胞Sonicator </td><td> bandelin </td><td> D - 1000 </td></tr><tr><td>荧光显微镜</td><td>荧光显微镜</td><td>徕卡</td><td> DMI6000B </td></tr><tr><td></td><td> 该软件的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>荧光显微镜</td> &lt;TD&gt; ASAF </td><td>徕卡</td><td></td></tr><tr><td>流式细胞仪</td><td> FCASDiva </td><td>屋宇署</td><td></td></tr><tr><td>流式细胞仪</td><td> FlowJo </td><td> treestar </td><td></td></tr></tbody></table>

single-cell analysis of bacillus subtilis biofilms using fluorescence microscopy and flow cytometry

几乎所有的细菌1-6生物膜的形成是一个普遍的属性。当细菌形成生物膜，包裹在细胞外基质，大多在7-10的其他因素所构成的蛋白质和胞外多糖，。包裹内生物膜的微生物群落，往往显示11-17专门的细胞不同亚群的分化。这些亚群共存，并常常表现出时间和空间内的生物膜18-21组织。 在模式生物的枯草芽孢杆菌的生物膜的形成需要专门的细胞不同亚群的分化。其中，亚矩阵生产商，负责产生和分泌的生物膜的细胞外基质是必不可少的生物膜形成11,19。因此，矩阵生产者的分化是生物膜形成的标志在 B 枯草。 我们使用荧光记者可视化和量化亚矩阵生产者B 的生物膜枯草 15,19,22-24。具体来说，我们观察到亚矩阵生产者自产外信号表面活性25存在区别。有趣的是，表面活性是由亚矩阵生产者15不同的专门细胞亚群。 我们已详列于本报告必要的技术方法，可视化和量化矩阵生产者和表面活性生产者在枯草杆菌生物膜亚群。要做到这一点，基质生产和生产表面活性所需的基因插入染色体B 的荧光记者枯草 。记者们表示，只有在一个专门的细胞亚群。然后，亚能监测用荧光显微镜和流式细胞仪（见图1）。 专门的细胞不同亚群内的细菌多细胞群落共存的事实给了我们一个不同的角度对原核生物的基因表达调控。该协议解决了这一实验现象和它可以很容易地适应任何其他的工作模式，以澄清在微生物群落的基本表型异质性的分子机制。

Watch this Scientific Journal Video about Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry at JoVE.com

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

微生物生物膜一般由专门的细胞不同亚群构成。这些亚群的单细胞分析需要使用荧光记者。在这里，我们描述了一个协议，以可视化和监视几个subpopulationswithin<em&gt; B枯草</em&gt;生物膜，用荧光显微镜和流式细胞仪。

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Research

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Immunology and Infection

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

科研

免疫与感染

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

热火预防应激在大鼠不利影响<em&gt;枯草芽孢杆菌</em&gt;应变

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

方法学研究<em&gt;乙。枯草</em&gt;生物膜作为模型的表征小分子抑制剂生物膜

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

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 ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...