弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件
Summary
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。
Abstract
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。
Protocol
1。制备的人包皮成纤维细胞（HFF）涂层的盖玻片井24孔组织培养板底部放置一个无菌的圆形玻璃盖玻片。 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。 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…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Bovine serum albumin | Sigma | A7906 | ||
| Dulbecco’s Modified Eagle Medium (DMEM) | Gibco | 11960-051 | ||
| Fetal Bovine Serum (FBS) | Atlanta Biologicals | S11150 | heat inactivate | |
| Rhodamine Dolichos biflorus agglutinin | Vector Laboratories | RL-1032 | ||
| Formaldehyde (16%) | Polysciences | 18814 | ||
| Glycine | Fisher Scientific | BP381-5 | ||
| HEPES | Fisher Scientific | BP310-1 | ||
| IFNγ | PeproTech | 315-05 | Store in single use aliquots | |
| L-glutamine (200mM) | Gibco | 25030 | ||
| Lipopolysaccharide (LPS) | Sigma | L4391-1MG | Store in single use aliquots | |
| Microscope Cover Glass | Fisher Scientific | 12-545-80 12CIR-1 | ||
| Penicillin-Streptomycin | Gibco | 15140 | ||
| RPMI medium 1640 powder (with L-glutamine, without bicarbonate) | Gibco | 31800-022 | ||
| Triton-X-100 | Fisher Scientific | BP151-500 | ||
| Trypsin-EDTA (0.25%) | Gibco | 25200 | ||
| VectaShield mounting media with DAPI | Vector Laboratories | H-1200 |
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Tags
Toxoplasma GondiiCyst Wall FormationBone Marrow-derived MacrophagesBradyzoite ConditionsObligate Intracellular ParasiteTachyzoiteEncysted FormSignaling ProcessChronic Stage Of InfectionCO2 StarvationHigh PH MediumInterferon Gamma (IFNγ)Dolichos Biflorus Agglutinin (DBA)Fluorescently Labeled DBAHuman Foreskin Fibroblasts (HFFs)Murine Bone Marrow-derived Macrophages (BMMs)Lipopolysaccharide (LPS)High PH Growth Medium
Cite This Article
Tobin, C., Pollard, A., Knoll, L. Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions. J. Vis. Exp. (42), e2091, doi:10.3791/2091 (2010).