细菌毒素引起的响应，用活细胞荧光显微镜的可视化
Summary
方法从重组净化胆固醇结合毒素链球菌溶血素O<em> E.大肠杆菌</em>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。
Abstract
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) | ||||||||||||||||||
| Ni-NTA agarose | Qiagen | 30210 | |||||||||||||||||||
| polymixin-agarose | Sigma | P1411-5ML | |||||||||||||||||||
| Zeba Desalt Spin col | Fisher | PI-89891 | |||||||||||||||||||
| sheep RBCs | Fisher | 50-415-688 | |||||||||||||||||||
| pBADgIII-SLO | N/A | N/A | see ref9 | ||||||||||||||||||
| Cy5 monoreactive dye | GE Healthcare | PA25001 | |||||||||||||||||||
| Fura2-AM | Life Technologies | F1221 | |||||||||||||||||||
| Calcein AM/ Ethidium homodimer | Life Technologies | L3224 | |||||||||||||||||||
| Anti-CD11c-APC | BD Biosciences | 550261 | |||||||||||||||||||
| collagen-coated glass-bottom dish | Mattek | P35GCol-1.5-10-C | |||||||||||||||||||
| femto-tip II | Fisher | E5242957000 | |||||||||||||||||||
| Microloader | Fisher | E5242956003 | |||||||||||||||||||
| dextran Alexa 555 | Life Technologies | D34679 | |||||||||||||||||||
| Injectman NI 2 | Eppendorf | 920000029 | |||||||||||||||||||
| FemtoJet | Eppendorf | 5247 000.013 | |||||||||||||||||||
Table 1. List and source of specific reagents and equipment needed. Specific equipment and reagents used in this protocol, along with company and catalogue number are listed. |
|||||||||||||||||||||
|
|||||||||||||||||||||
Table 2. List of buffers used in this protocol. The buffers used, their composition and the first step at which they are used in the protocol are listed. |
References
- Walev, I. Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O. Proc. Natl. Acad. Sci. U.S.A. 98, 3185-3190 (2001).
- Walev, I. Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2. J. Immunol. 164, 5120-5124 (2000).
- Scolding, N. J. Vesicular removal by oligodendrocytes of membrane attack complexes formed by activated complement. Nature. 339, 620-622 (1989).
- Keyel, P. A. Streptolysin O clearance through sequestration into blebs that bud passively from the plasma membrane. J. Cell. Sci. 124, 2414-2423 (2011).
- MacKenzie, A. Rapid secretion of interleukin-1beta by microvesicle shedding. Immunity. 15, 825-835 (2001).
- Qu, Y., Franchi, L., Nunez, G., Dubyak, G. R. Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages. J. Immunol. 179, 1913-1925 (2007).
- Andrei, C. Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes. Proc. Natl. Acad. Sci. U.S.A. 101, 9745-9750 (2004).
- Franchi, L., Eigenbrod, T., Munoz-Planillo, R., Nunez, G. The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis. Nat. Immunol. 10, 241-247 (2009).
- Magassa, N., Chandrasekaran, S., Caparon, M. G. Streptococcus pyogenes cytolysin-mediated translocation does not require pore formation by streptolysin O. EMBO Rep. 11, 400-405 (2010).
- Pinkney, M., Beachey, E., Kehoe, M. The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity. Infect. Immun. 57, 2553-2558 (1989).
- Watkins, S. C., Salter, R. D. Functional connectivity between immune cells mediated by tunneling nanotubules. Immunity. 23, 309-318 (2005).
- McNeil, P. L., Vogel, S. S., Miyake, K., Terasaki, M. Patching plasma membrane disruptions with cytoplasmic membrane. J. Cell. Sci. 113, 1891-1902 (2000).
- Shannon, J. G., Ross, C. L., Koehler, T. M., Rest, R. F. Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin. Infect. Immun. 71, 3183-3189 (2003).
