|
Author:
Date:
2014-10-20
[Abstract] Gram negative bacterial pathogens, such as Shigella flexneri, which possess a Type Three Secretion System (T3SS), are able to transfer bacterial proteins, dubbed translocators and effectors, from their cytoplasm into the cytoplasm of their host cells using a syringe like needle complex. For Shigella, it has been shown that during cellular invasion, the intrabacterial pool of translocators and effectors is completely depleted upon activation of the TTS Apparatus and is then progressively replenished while bacteria remain inside host cells. Replenishment of effectors allows for cell-to-cell spreading events, which also necessitate reactivation of the T3SA, and lead to another round of depletion of intrabacterial effector stores. To understand the state of individual ...
[摘要] 具有三型分泌系统（T3SS）的革兰氏阴性细菌病原体如灵芝氏菌能够将细菌蛋白质，配位的易位蛋白和效应子从其细胞质转移到其宿主细胞的细胞质中使用注射器如针复合物。对于志贺氏菌（Shigella），已经表明在细胞侵袭过程中，转位子和效应子的细菌池在TTS装置激活时完全耗尽，然后逐渐补充，同时细菌保留在宿主细胞内。补充效应物允许细胞间的扩散事件，这也需要T3SA的再活化，并导致细胞内效应子库的另一轮消耗。为了理解感染期间单个细胞内细菌的状态，因此有兴趣的是能够定位和评估细菌和分泌的易位蛋白和效应子池的相对数量。我们最近改编了基于EDTA和溶菌酶的方法以透化宿主细胞内存在的细菌的细胞壁，以标记尖端蛋白IpaD和易位蛋白IpaB和IpaC的细菌池。在这里，我们详细描述执行连续标记细菌和分泌池的协议。这种方法理论上可扩展到由其他分泌系统和其他细菌病原体分泌的毒力因子。
|
|
Author:
Date:
2013-11-20
[Abstract] Flow cytometry, a standard technique used for quantitative analysis of isolated cells, is routinely employed by immunologists and oncologists to study DNA content, protein expression, and other functional parameters in blood and tumor cells. Unfortunately, the use of this technique by neurobiologists has been hampered by the complexity of the nervous system, whose constituting cells can hardly be dissociated to obtain samples of sufficient quality. We have developed a simplified and quick method to purify and immunolabel cell nuclei with high sensitivity and low background. Our protocol allows the discrimination of single nuclei from doublets and larger aggregates, obtaining low coefficients of variation for cell cycle analysis with propidium iodide. In addition, due to the reduced sample ...
[摘要] Flow cytometry, a standard technique used for quantitative analysis of isolated cells, is routinely employed by immunologists and oncologists to study DNA content, protein expression, and other functional parameters in blood and tumor cells. Unfortunately, the use of this technique by neurobiologists has been hampered by the complexity of the nervous system, whose constituting cells can hardly be dissociated to obtain samples of sufficient quality. We have developed a simplified and quick method to purify and immunolabel cell nuclei with high sensitivity and low background. Our protocol ...
|