Name: establishment of an in vitro system to study intracellular behavior of candida glabrata in human thp-1 macrophages
Uploaded: 2013-12-10T20:15:00
Description: Watch this Scientific Journal Video about Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages at JoVE.com

这项工作得到了创新的年轻生物技术专家奖BT/BI/12/040/2005和BT/PR13289/BRB/10/745/2009赠款生物技术，印度和中心的DNA指纹图谱和诊断核心资金的政府部门的支持，海得拉巴。 MNR和GB是初中和科学与工业研究理事会对追求马尼帕尔大学博士学位的高级研究奖学金的受助人。 SB是初中和生物技术部高级研究奖学金朝着追求马尼帕尔大学的博士学位获得者。

建议在执行C。光滑念珠菌感染的实验与生物安全防护水平的2实验室（BSL-2）。 <ol><li>制备THP-1巨噬细胞单层。 </li><li> C.编制光滑念珠菌细胞悬液。 </li><li> THP-1巨噬细胞C的感染光滑的细胞。 </li><li>测量通过集落形成单位测定吞噬率和细胞内复制。 </li><li>监控用共聚焦激光扫描显微镜细胞内复制。 </li></ol> 1。 THP-1巨噬细胞单分子膜的制备<ol>...

<ol>
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先天免疫系统中起着的机会性真菌感染的控制中起重要作用。巨噬细胞促进食入​​和破坏病原真菌的抗真菌防御。因此，所需要的生存和/或对抗巨噬细胞的抗菌功能的因素，阐明将增进我们对真菌毒力的战略认识。在这方面，我们已经建立了使用来自人单核细胞系THP-1巨噬细胞以表征一个人的机会性真菌病原体C的相互作用在体外细胞培养模型系统光滑念珠菌与宿主吞噬细胞...

念珠菌是免疫功能低下患者1危及生命的侵袭性真菌感染的主要原因。 光滑念珠菌 ，一个新兴的院内病原体，是从重症监护病房的患者取决于地理位置1-3第二个或第三个最常见的孤立念珠菌。系统发育，C.光滑念珠菌 ，单倍体芽殖酵母，更密切相关的非致病模型酿酒酵母不是致病念珠菌。包括C。白色念珠菌 4。与此相一致，C。光滑缺少一些关键的真菌毒力性状，包括交配，分泌的蛋白水解活性和形态可塑性4-5。 虽然C。光滑不形成菌丝，它可以生存和复制在小鼠和人类巨噬细胞6-8这表明它已经开发了独特的发病机制。有限的信息，请访问有关的策略，C.采用光滑的生存贫营养的巨噬细胞内环境，抵制氧化和挂载宿主免疫细胞5非氧化宿主反应。一个与此相关巨噬细胞模型系统是一个先决条件划定C的相互作用光滑念珠菌与宿主吞噬细胞通过功能基因组学和蛋白质组学的方法。外周血单核细胞（PBMC）和人及鼠源的骨髓衍生的巨噬细胞（BMDMs），分别刚才被用来研究C的相互作用光滑念珠菌与宿主免疫细胞7,9。然而，难以获得外周血单个核细胞和BMDMs，他们有限的生命跨度和不同哺乳动物捐助者之间的内在差异限制了这些细胞的利用率多才多艺的模型系统。 在这里，我们描述了一种建立在体外系统研究intracellul的C的AR行为光滑念珠菌细胞从人单核细胞系THP-1巨噬细胞。本协议的总体目标是制定可以很容易地操纵研究宿主真菌病原体相互作用的不同方面简单，价格低廉，快速，重现性细胞培养模型系统。 THP-1细胞先前已用于解密对广范围的病原体，包括细菌，病毒，真菌和10-12的宿主免疫应答。单核细胞THP-1细胞易于维护，并且可以区分，在佛波酯治疗，巨噬细胞它模仿人类单核细胞源性巨噬细胞和表达适当的巨噬细胞标记物13。 THP-1巨噬细胞模型系统的主要优点是易于使用性和缺乏先进的设备要求。 这里介绍的协议是很容易适应学习其他人的真菌病原体与居屋的相互作用吨的免疫细胞。当前的过程也可以用来识别毒力因子的感兴趣使用高通量突变体屏幕上的病原体。这证明型概念例证的成功使用THP-1培养模型系统来识别一组在人类巨噬细胞8所需的光滑念珠菌生存的56个基因。

<table border="0" cellpadding="0" cellspacing="0" width="596">
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	<tbody>
		<tr height="39">
			<td height="39" style="height:40px;width:163px;">THP-1</td>
			<td style="width:105px;">American Type Culture Collection</td>
			<td style="width:85px;">TIB 202</td>
			<td style="width:243px;">Human acute monocytic leukemia cell line</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">RPMI-1640</td>
			<td style="width:105px;">Hyclone</td>
			<td style="width:85px;">SH30096.01</td>
			<td style="width:243px;">For maintaining THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Phorbol 12-myristate 13-acetate</td>
			<td style="width:105px;">Sigma-Aldrich</td>
			<td style="width:85px;">P 8139</td>
			<td style="width:243px;">Caution: Hazardous</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">YPD&#160;</td>
			<td style="width:105px;">BD-Difco</td>
			<td style="width:85px;">242710</td>
			<td style="width:243px;">For growing Candida glabrata cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Formaldehyde</td>
			<td style="width:105px;">Sigma-Aldrich</td>
			<td style="width:85px;">F8775</td>
			<td style="width:243px;">For fixation of C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Phosphate buffered saline (PBS)</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl,&#160; pH 7.4) for washes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Saline-sodium citrate (SSC)&#160;</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (3 M NaCl, 0.3 M sodium citrate for 20x concentration) for washes</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Prehybridization buffer</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (50% formamide, 5x Denhardt&#8217;s solution, 5x SSC, 1% SDS) for hybridization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">VECTASHIELD mounting medium</td>
			<td style="width:105px;">Vector Labs</td>
			<td style="width:85px;">H-1200</td>
			<td style="width:243px;">For mounting slides for confocal microscopy</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">32P-labeled &#945;-dCTP</td>
			<td style="width:105px;">JONAKI-BARC</td>
			<td style="width:85px;">LCP-102</td>
			<td style="width:243px;">For radiolabeling of signature tags</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">100 mm tissue culture dishes</td>
			<td style="width:105px;">Corning</td>
			<td style="width:85px;">430167</td>
			<td style="width:243px;">To culture THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">24-well tissue culture plate</td>
			<td style="width:105px;">Corning</td>
			<td style="width:85px;">3527</td>
			<td style="width:243px;">To perform C. glabrata infection studies in THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">4-chamber tissue culture-treated glass slide</td>
			<td style="width:105px;">BD Falcon</td>
			<td style="width:85px;">REF354104</td>
			<td style="width:243px;">To image C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Hemocytometer</td>
			<td style="width:105px;">Rohem India</td>
			<td style="width:85px;"></td>
			<td style="width:243px;">For enumeration of cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Table top microcentrifuge</td>
			<td style="width:105px;">Beckman Coulter</td>
			<td style="width:85px;">Microfuge 18</td>
			<td style="width:243px;">For spinning down cells in microtubes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Table top centrifuge</td>
			<td style="width:105px;">Remi</td>
			<td style="width:85px;">R-8C</td>
			<td style="width:243px;">For spinning down cells in 15 ml tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Spectrophotometer</td>
			<td style="width:105px;">Amersham Biosciences</td>
			<td style="width:85px;">Ultraspec 10</td>
			<td style="width:243px;">To monitor absorbance of yeast cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Plate incubator</td>
			<td style="width:105px;">Labtech</td>
			<td style="width:85px;">Refrigerated</td>
			<td style="width:243px;">To grow C. glabrata cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Shaker incubator</td>
			<td style="width:105px;">New Brunswick</td>
			<td style="width:85px;">Innova 43</td>
			<td style="width:243px;">To grow C. glabrata cells</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Water jacketed CO2&#160; incubator</td>
			<td style="width:105px;">Thermo Electron Corporation</td>
			<td style="width:85px;">Forma series 2</td>
			<td style="width:243px;">To culture THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Confocal microscope</td>
			<td style="width:105px;">Carl Ziess</td>
			<td style="width:85px;">Ziess LSM 510 meta&#160;</td>
			<td style="width:243px;">To observe C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Compound microscope</td>
			<td style="width:105px;">Olympus</td>
			<td style="width:85px;">CKX 41</td>
			<td style="width:243px;">To observe C. glabrata and THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">PCR machine</td>
			<td style="width:105px;">BioRad&#160;</td>
			<td style="width:85px;">DNA Engine</td>
			<td style="width:243px;">To amplify unique tags from input and output genomic DNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Hybridization oven</td>
			<td style="width:105px;">Labnet</td>
			<td style="width:85px;">Problot 12S</td>
			<td style="width:243px;">For hybridization&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">PhosphorImager</td>
			<td style="width:105px;">Fujifilm</td>
			<td style="width:85px;">FLA-9000</td>
			<td style="width:243px;">For scanning hybridized membranes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Thermomixer</td>
			<td style="width:105px;">Eppendorf</td>
			<td style="width:85px;">Thermomixer Comfort</td>
			<td style="width:243px;">For denaturation of radiolabeled signature tags</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:163px;">Gel documentation unit</td>
			<td style="width:105px;">Alphainnontech</td>
			<td style="width:85px;">Alphaimager</td>
			<td style="width:243px;">To visualize ethidium bromide-stained DNA in agarose gels</td>
		</tr>
	</tbody>
</table>

establishment of an in vitro system to study intracellular behavior of candida glabrata in human thp-1 macrophages

一种细胞培养模型系统中，如果主机环境条件密切模仿，可以作为一种廉价的，可再现的和容易可操纵替代动物模型系统中的微生物病原体感染的特定步骤的研究。一个人单核细胞株THP-1其中，经佛波酯治疗，分化为巨噬细胞，以前被用来研究的许多细胞内的病原体包括结核杆菌毒力的战略。在这里，我们讨论的协议制定的体外细胞培养模型使用THP-1巨噬细胞中描绘的人的机会性病原体酵母光滑念珠菌与宿主吞噬细胞的相互作用的系统。该模型系统结构简单，速度快，适合于高通量突变屏幕，并且不需要复杂的设备。一个典型的THP-1巨噬细胞感染实验大约需要24小时一个额外的24-48小时，使细胞内收回酵母生长在营养丰富的培养基进行菌落形成单位为基础的可行性分析。像其他的体外模型系统中，这种方法的一个可能的限制是难以外推得到的一个高度复杂的免疫细胞的电路存在于人类宿主的结果。然而，尽管这样，目前的协议是要阐明病原真菌可以采用回避/抵抗抗生素的反应和生存，适应和增殖的宿主免疫细胞的营养环境恶劣的策略非常有用。

PMA处理的THP-1巨噬细胞C.感染分析光滑念珠菌的野生型（wt）细胞中发现， 重量细胞，巨噬细胞在2小时后共同培养的速率的55-65％，吞噬。此外，C。光滑念珠菌细胞能够由THP-1巨噬细胞的抗杀并进行了适度的5 -至与THP-1巨噬细胞的共培养8的24小时后的7倍增加的CFUs。野生型细胞，转化有表达GFP的质粒，在THP-1巨噬细胞内的复制，也验证了用共聚焦荧光显微镜...

Watch this Scientific Journal Video about Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages at JoVE.com

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

当前文章概述了一个协议来建立体外细胞培养模型系统来研究一种兼人体细胞内的真菌病原体光滑念珠菌与人体巨噬细胞将是一个有用的工具，以增进我们对真菌毒力机制知识的互动。

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Research

JoVE Journal

Immunology and Infection

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

科研

免疫与感染

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Study of Phagolysosome Biogenesis in Live Macrophages

在现场研究巨噬细胞吞噬溶酶体生源

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

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 ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

具有体外转录mRNA的初级巨噬细胞的高效转染

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...