Al adjuvants can be tracked in viable cells by lumogallion staining
Al adjuvants can be tracked in viable cells by lumogallion staining
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DOI：
10.1016/j.jim.2015.04.008
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发表时间：
2015-07-01
影响因子：
2.2
通讯作者：
Eriksson, Hakan
中科院分区：
文献类型：
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作者：
Mile, Irene;Svensson, Andreas;Eriksson, Hakan
尽管铝盐在临床疫苗中已经使用了几十年，但铝盐佐剂作用背后的机制却知之甚少。在水环境和近中性的pH条件下，铝盐形成颗粒聚集体，对于缺乏有关机制的信息，一个合理的解释可能是缺乏有效的方法来跟踪吞噬的铝佐剂，从而确定佐剂在细胞内的位置。在这篇文章中，我们想要报告使用Lumogalion染色来检测活细胞内吞噬的铝佐剂。在培养上清液中加入微摩尔浓度的虫草，可使吞噬铝佐剂的细胞发出强烈的荧光信号。铝佐剂在细胞质中呈斑点状，通过共聚焦显微镜和与荧光探针的共染色，在光谱的远红区，可以在一定程度上确定铝佐剂定位于酸性囊泡，即溶酶体。细胞内铝佐剂的保存和检测不仅通过隆起扩散到细胞质中来实现，从而突出佐剂的存在，而且还通过在与细胞孵育之前对铝佐剂进行预染色来实现。铝佐剂的预染色导致明亮的荧光颗粒聚集体保持荧光数周，并且在广泛洗涤或与细胞孵育时仅有轻微的荧光减弱。氢氧化铝和羟基磷酸铝佐剂是临床疫苗中最常用的两种铝佐剂，它们都能被包被蛋白预先染色，并在与吞噬细胞孵育后容易被追踪到细胞内。利用包被蛋白获得活细胞将是研究铝佐剂将抗原提呈细胞分化为炎症细胞的机制的有用方法。将获得有关吞噬小体途径和吞噬小体内部事件的信息，从而可以解决吞噬细胞中铝佐剂的最终命运。(C)2015爱思唯尔B.V.保留所有权利。
The mechanism behind the adjuvant effect of aluminum salts is poorly understood notwithstanding that aluminum salts have been used for decades in clinical vaccines. In an aqueous environment and at a nearly neutral pH, the aluminum salts form particulate aggregates, and one plausible explanation of the lack of information regarding the mechanisms could be the absence of an efficient method of tracking phagocytosed aluminum adjuvants and thereby the intracellular location of the adjuvant.In this paper, we want to report upon the use of lumogallion staining enabling the detection of phagocytosed aluminum adjuvants inside viable cells. Including micromolar concentrations of lumogallion in the culture medium resulted in a strong fluorescence signal from cells that had phagocytosed the aluminum adjuvant. The fluorescence appeared as spots in the cytoplasm and by confocal microscopy and co-staining with probes presenting fluorescence in the far-red region of the spectrum, aluminum adjuvants could to a certain extent be identified as localized in acidic vesicles, i.e., lysosomes.Staining and detection of intracellular aluminum adjuvants was achieved not only by diffusion of lumogallion into the cytoplasm, thereby highlighting the presence of the adjuvant, but also by pre-staining the aluminum adjuvant prior to incubation with cells. Pre-staining of aluminum adjuvants resulted in bright fluorescent particulate aggregates that remained fluorescent for weeks and with only a minor reduction of fluorescence upon extensive washing or incubation with cells. Both aluminum oxyhydroxide and aluminum hydroxyphosphate, two of the most commonly used aluminum adjuvants in clinical vaccines, could be pre-stained with lumogallion and were easily tracked intracellularly after incubation with phagocytosing cells.Staining of viable cells using lumogallion will be a useful method in investigations of the mechanisms behind aluminum adjuvants' differentiation of antigen-presenting cells into inflammatory cells. Information will be gained regarding the phagosomal pathways and the events inside the phagosomes, and thereby the ultimate fate of phagocytosed aluminum adjuvants could be resolved. (C) 2015 Elsevier B.V. All rights reserved.