Name: visualization of bacterial toxin induced responses using live cell fluorescence microscopy
Uploaded: 2012-10-01T13:00:00
Description: Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

作者想，感谢理查德休息的SLO质粒和乔纳森·弗兰克斯提供技术援助的慷慨馈赠的厚礼，anthrolysin O，迈克尔Caparon。这项工作是由NIH拨款T32CA82084（PAK），和R01AI072083（RDS）。

1。纯化的链球菌溶血素O（SLO） <ol><li>接种20毫升过夜培养BL21 GOLD细胞含有pBADgIII-SLOhis质粒9到0.5升的LB肉汤，并加入500微升50毫克/毫升氨苄青霉素。在225的转速在37℃下振荡培养直至OD 600，通常为0.6〜1.5小时。在10,000 xg离心5分钟，离心1 ml细菌（考虑T 0），溶解沉淀于140μL1×SDS上样缓冲液/ 1 OD和超声剪切DNA的蛋白质纯度分析。 </li><li>诱导细菌与加入5毫升20％的阿拉伯糖文?...

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这里描述的技术允许检查细菌毒素的免疫细胞的反应。最关键的步骤是处理和剂量的毒素。毒素活性可以是非常可变之间，甚至在不同的等分试样的相同的制备，由于其脆弱性。因此，有必要对引用单元格的行或红细胞的毒素测试各等份，或使用毒素梯度的。毒素梯度，交付微量移液器，让毒素引起的活动时必须遵守的实时全方位的，但不适合本身的生化分析。 微管交货的?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> Ni-NTA琼脂糖</td><td> Qiagen公司</td><td> 30210 </td><td></td></tr><tr><td>多粘菌素琼脂糖</td><td>西格玛</td><td> P1411-5ML </td><td></td></tr><tr><td>泽巴脱盐旋转山坳</td><td>费舍尔</td><td> PI-89891 </td><td></td></tr><tr><td>绵羊红细胞</td><td>费舍尔</td><td> 50-415-688 </td><td></td></tr><tr><td> pBADgIII-SLO </td><td> N / A </td><td> N / A </td><td>见文献9 </td></tr><tr><td> Cy5的monoreactive染料</td><td> GE医疗集团</td><td> PA25001 </td><td></td></tr><tr><td> Fura2-AM </td><td> Life Technologies公司</td><td> F1221 </td><td></td></tr><tr><td>钙黄绿素AM / EThidium二聚体</td><td> Life Technologies公司</td><td> L3224 </td><td></td></tr><tr><td>抗CD11c-APC </td><td> BD Biosciences公司</td><td> 550261 </td><td></td></tr><tr><td>胶原涂层的玻璃底培养皿</td><td> Mattek </td><td> P35GCol-1.5-10-C </td><td></td></tr><tr><td>毫微微尖II </td><td>费舍尔</td><td> E5242957000 </td><td></td></tr><tr><td> Microloader </td><td>费舍尔</td><td> E5242956003 </td><td></td></tr><tr><td>右旋糖酐Alexa的555 </td><td> Life Technologies公司</td><td> D34679 </td><td></td></tr><tr><td> Injectman NI 2 </td><td> Eppendorf公司</td><td> 920000029 </td><td></td></tr><tr><td> FemtoJet </td><td> Eppendorf公司</td><td> 5247 000.013 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表1中。列表和源的具体试剂和equipm耳鼻喉科需要。在该协议中使用的特殊设备和试剂，以及公司和产品目录号上市。 </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 缓冲区 </td><td> 组成 </td><td> 步骤使用 </td></tr><tr><td>裂解/清洗</td><td>的50mM的NaH 2 PO 4的 300mM的NaCl 的10mM咪唑，pH 8.0 </td><td> 1.4 </td></tr><tr><td>三/盐</td><td>的50mM Tris，pH值8.0 300mM的NaCl </td><td> 1.7 </td></tr><tr><td>洗脱缓冲液</td><td>的50mM Tris，pH值8.0 300mM的NaCl 250 mM咪唑</td><td> 1.8 </td></tr><tr><td> RBC分析缓冲液</td><td> 0.3％BSA 2毫米氯化钙 的10mM HEPES，pH值7.4 </td><td> 2.1 </td></tr><tr><td>缓冲R / B </td><td>的RPMI细胞培养基 2毫米氯化钙 0.5％BSA的</td><tD&gt; 3.1 </td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td> 表2列出在本协议中所使用的缓冲区。使用的缓冲液，它们的组合物和它们在协议中使用的第一个步骤中列出。 </td></tr></tbody></table>

visualization of bacterial toxin induced responses using live cell fluorescence microscopy

细菌毒素结合到膜中的胆固醇，细胞内容物的泄漏，和来自外部环境的材料涌入允许形成细孔。从这个侮辱，这需要积极的膜修复过程的细胞可以恢复，否则死取决于毒素的暴露量和细胞类型1。此外，这些毒素诱导强烈的炎症反应，在受感染的宿主中，通过激活免疫细胞，包括巨噬细胞，从而产生的促炎性细胞因子的阵列2。许多革兰氏阳性细菌产生的胆固醇结合的毒素，这已被证明有助于其毒力，很大程度上是未知的机制。 这些毒素的细胞暴露在质膜的形态学改变包括其螯合成富含胆固醇的表面突起，它可以脱落到细胞外空间，提示的特性的细胞防御机制3,4。此过程发生的代谢活动的情况下，在所有细胞中，可以可视化后使用EM化学固定4。在免疫细胞如巨噬细胞介导炎症的毒素暴露，引起膜泡的建议包含细胞因子IL-1家族，可能是负责脱落毒素和传播这些促炎性细胞因子5,6,7。 IL-1β的释放之间的链接，以及一种特定类型的细胞死亡，称为pyroptosis已建议，因为二者都是caspase-1的依赖过程8。进行排序，满分该巨噬细胞反应的复杂性，其中包括毒素结合，脱落的膜性囊泡，细胞因子的释放，和潜在的细胞死亡，我们已经开发了标记技术和荧光显微镜观察的方法，使毒素的细胞间的相互作用的实时可视化的，包括测量功能障碍和死亡（ 图1）。使用活细胞成像是必要的，因为其他技术的限制。生化方法不能解决发生在单个细胞的效应，而流式细胞仪没有提供高分辨率，单个细胞的实时可视化。这里描述的方法可以适用于由其他涉及复杂的细胞的表型变化的刺激诱导的反应的动力学分析。

通常为10 7 -10 8 U / ml的SLO可以得到4毫克/毫升的蛋白浓度。毒素细胞裂解所需的量的细胞类型而变化，但通常为125-500 U / ml的SLO（ 图2B）。如巨噬细胞的细胞类型可以更耐（4000 U /毫升），但其他人（特别是T细胞系）更为敏感。这些敏感性与市售SLO。毒素活性降低大约2倍的与各冻结 - 解冻，因此溶血检测与毒素每批或解冻需要验证毒素活性。毒素是在的情况下，含胆固...

Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

方法从重组净化胆固醇结合毒素链球菌溶血素O<em&gt; E.大肠杆菌</em&gt;和可视化的生活真核细胞毒素结合进行了阐述。本地化的交付毒素诱导靶细胞，揭示新的毒素生物学方面的快速而复杂的变化。

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

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