Name: toxoplasma gondii cyst wall formation in activated bone marrow-derived macrophages and bradyzoite conditions
Uploaded: 2010-08-12T17:30:00
Description: Watch this Scientific Journal Video about Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions at JoVE.com

这项工作是由威斯康星 - 麦迪逊赫尔曼一夏皮罗杰出研究生奖学金（CMT），美国国立卫生研究院奖AI072817（AMP）和美国心脏协会奖0840059N（LJK）大学的支持。

1。制备的人包皮成纤维细胞（HFF）涂层的盖玻片<ol><li>井24孔组织培养板底部放置一个无菌的圆形玻璃盖玻片。 </li><li>收获HFFS从一个融合150厘米2烧瓶，冲洗烧瓶中两次，用1X PBS和添加2.5毫升0.025％胰蛋白酶EDTA 。在37 ° C为5-10分钟烧瓶中。 </li><li>攻方对手掌的容量瓶，可以帮助从烧瓶中分离的细胞。一旦细胞有从烧瓶中公布，新增150 HFF中等毫升（贝科的修改鹰中等[]与10％胎牛血清的DME...

<ol>
	<li>Tomida, M., Yamamoto-Yamaguchi, Y., Hozumi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+a+factor+inducing+differentiation+of+mouse+myeloid+leukemic+M1+cells+from+conditioned+medium+of+mouse+fibroblast+L929+cells.">Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells.</a> J Biol Chem. 259 (17), 10978-10980 (1984).</li><li>Matrajt, M., Donald, R. G., Singh, U., Roos, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+differentiation+mutants+in+the+protozoan+parasite+Toxoplasma+gondii.">Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii.</a> Mol Microbiol. 44, 735-747 (2002).</li><li>Singh, U., Brewer, J. L., Boothroyd, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+tachyzoite+to+bradyzoite+differentiation+mutants+in+Toxoplasma+gondii+reveals+a+hierarchy+of+gene+induction.">Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction.</a> Mol Microbiol. 44, 721-733 (2002).</li><li>J, P., Knoll, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Toxoplasma+gondii+development+mutants+identifies+a+potential+proteophosphogylcan+that+enhances+cyst+wall+formation.+Molecular+and+Biochemical+Parasitology.">Isolation of Toxoplasma gondii development mutants identifies a potential proteophosphogylcan that enhances cyst wall formation. Molecular and Biochemical Parasitology.</a> 169 (2), 120-123 (2010).</li><li>Bohne, W., Heesemann, J., Gross, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Bradyzoite-Specific+Toxoplasma+gondii+Antigens+in+Gamma+Interferon-Treated+Mouse+Macrophages.">Induction of Bradyzoite-Specific Toxoplasma gondii Antigens in Gamma Interferon-Treated Mouse Macrophages.</a> Infection and Immunity. 61 (3), 1141-1145 (1993).</li><li>Ibrahim, H. M., Bannai, H., Xuan, X., Nishikawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii+Cyclophilin+18-Mediated+Production+of+Nitric+Oxide+Induces+Bradyzoite+Conversion+in+a+CCR5-Dependent+Manner.">Toxoplasma gondii Cyclophilin 18-Mediated Production of Nitric Oxide Induces Bradyzoite Conversion in a CCR5-Dependent Manner.</a> Infection and Immunity. 77 (9), 3686-3695 (2009).</li><li>Lyons, R. E., McLeod, R., Roberts, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii+tachyzoite-bradyzoite+interconversion.">Toxoplasma gondii tachyzoite-bradyzoite interconversion.</a> Trends in Parasitology. 18 (5), 198-201 (2002).</li><li>Berghaus, L. J., Moore, J. N., Hurley, D. J., Vandenplas, M. L., Fortes, B. P., Wolfert, M. A., Boons, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+responses+of+primary+murine+macrophage-lineage+cells+and+RAW+264.7+cells+to+ligands+of+Toll-like+receptors+2,+3,+and+4.">Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors 2, 3, and 4.</a> Comp Immunol Microbiol Infect Dis. , (2009).</li><li>Mordue, D. G., Scott-Weathers, C. F., Tobin, C. M., Knoll, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+patatin-like+protein+protects+Toxoplasma+gondii+from+degradation+in+activated+macrophages.">A patatin-like protein protects Toxoplasma gondii from degradation in activated macrophages.</a> Mol Microbiol. 63 (2), 482-496 (2007).</li><li>Bohne, W., Heesemann, J., Gross, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+replication+of+Toxoplasma+gondii+is+necessary+for+induction+of+bradyzoite-specific+antigens:+a+possible+role+for+nitric+oxide+in+triggering+stage+conversion.">Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: a possible role for nitric oxide in triggering stage conversion.</a> Infection and Immunity. 62 (5), 1761-1777 (1994).</li></ol>

虽然bradyzoite发展的机制尚不完全清楚，T的分子遗传分析弓形虫在组织培养的阶段转换，导致在 2,3,4 bradyzoite囊肿形成有关的基因的发现。也导致了一些bradyzoite标记其他长期胁迫条件下，包括在激活的巨噬细胞的增长 5,6的表达观察分析。上面的方法描述了如何成长T 。 弓形虫诱导发展一个DBA的积极骨髓细胞和HFFS囊肿壁结构。这些结果突出，囊肿发展途?...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7906</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Modified Eagle Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11960-051</td>
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<td>Fetal Bovine Serum (FBS)</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11150</td>
<td>heat inactivate </td>
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<td>Rhodamine Dolichos biflorus agglutinin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>RL-1032</td>
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<td>Polysciences</td>
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<td>BP381-5</td>
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<td>Fisher Scientific</td>
<td>BP310-1</td>
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<td>PeproTech</td>
<td>315-05</td>
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<td>&nbsp;</td>
<td>Gibco</td>
<td>25030</td>
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<td>&nbsp;</td>
<td>Sigma</td>
<td>L4391-1MG</td>
<td>Store in single use aliquots</td>
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<td>Microscope Cover Glass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-80 12CIR-1</td>
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<td>15140</td>
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<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
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toxoplasma gondii cyst wall formation in activated bone marrow-derived macrophages and bradyzoite conditions

弓形虫是一种专性细胞内寄生虫能侵入任何温血动物有核细胞。在感染，T。弓形虫传播作为一种快速复制的形式被称为速殖子。速殖子转换成一个缓慢增长的囊称为一个信号的过程，是没有很好的特点bradyzoite的形式。 bradyzoite囊肿内的动物，被发现在中枢神经系统和肌肉组织和代表的慢性感染阶段。可以进行模拟转换到bradyzoites在组织培养中的CO 2饥饿，使用高pH值，或增加干扰素γ（IFNγ）的媒介。 Bradyzoites的特点是存在一个囊肿壁，其中的凝集素的Dolichos biflorus凝集素（DBA）结合。荧光标记的DBA是用于可视化的囊壁在人包皮成纤维细胞（HFFS）已暴露在低CO 2和高pH值介质中生长的寄生虫。同样，寄生虫居住在小鼠骨骨髓来源的巨噬细胞（BMMs）显示囊肿壁由DBA的检测γ干扰素和脂多糖（LPS）激活后的楼宇管理维修综合计划“。此协议将演示如何诱导T 的转换弓形虫到低的CO 2和楼宇管理维修综合计划“激活使用高pH值增长介质的bradyzoites。宿主细胞培养上盖玻片，与速殖子感染，要么与γ干扰素和脂多糖（BMMs）此外激活或暴露于高pH值生长介质三天（HFFS）。宿主细胞的感染完成后，将固定，通透，并阻止。囊壁将使用荧光显微镜罗丹明DBA的可视化。

Watch this Scientific Journal Video about Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions at JoVE.com

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

弓形虫囊肿的形式，在应对环境压力，可在组织培养模式模仿的转换。本视频演示了技术审查囊肿壁的形成，激活骨髓来源的巨噬细胞或改变成纤维细胞生长介质的pH值。

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

Research

JoVE Journal

Immunology and Infection

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

取得高纯度弓形虫</em一个不连续的氯化铯梯度>卵囊

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

科研

免疫与感染

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

遗传筛选隔离弓形虫宿主细胞的出口突变

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

遗传操纵<em&gt;Δku80</em&gt;株功能基因组分析<em&gt;弓形虫</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Bone Marrow-derived Macrophage Production

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

正向遗传学屏风使用巨噬细胞识别<em&gt;弓形虫</em&gt;基因的重要的抗IFN-γ依赖性细胞免疫自主

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

骨髓来源巨噬细胞的分离和 体外 培养用于NO-氧化还原生物学研究

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...