Collins Andrew J, Nyholm Spencer V
Department of Molecular and Cell Biology, University of Connecticut, Storrs, USA.
J Vis Exp. 2010 Feb 11(36):1714. doi: 10.3791/1714.
Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host's innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy.
近年来，有关免疫系统在介导有益细菌与其宿主之间分子信号传导中作用的研究，为我们理解真核生物与其微生物群的共同进化做出了重大贡献。夏威夷短尾乌贼（Euprymna scolopes）与发光细菌费氏弧菌（Vibrio fischeri）之间的共生关系，已被用作一个模型系统，用于理解有益细菌对动物发育的影响。最近的研究表明，巨噬细胞样血细胞是乌贼宿主先天免疫系统的唯一细胞成分，可能在介导这种共生关系的建立和维持中发挥重要作用。本实验方案将演示如何从夏威夷短尾乌贼中获取血细胞，然后将这些细胞用于细菌结合试验。首先对成年乌贼进行麻醉，然后用注射器从主要的头部血管收集血淋巴。提取的血淋巴中所含的宿主血细胞，被粘附在有腔的盖玻片上，然后暴露于绿色荧光蛋白标记的共生费氏弧菌和非共生哈维氏弧菌（Vibrio harveyi）中。血细胞用荧光染料（Cell Tracker Orange，英杰公司）复染，然后用荧光显微镜观察。
