Phagocytic activity of Limulus polyphemus amebocytes in vitro

Phagocytosis of invading microorganisms is a fundamental component of innate immunity. The Atlantic horseshoe crab, Limulus polyphemus, possesses a single immune cell type, the granular amebocyte. Amebocytes release a repertoire of potent immune effectors in the presence of pathogens, and function in hemostasis. In contrast to other arthropod immunocytes, the properties of amebocyte phagocytosis remain poorly characterised, restricted by the technical challenges associated with handling these labile cells. We have addressed these challenges and observed the internalisation of microbial and synthetic targets by amebocytes in vitro. Confirmation of target internalisation was achieved using a combination of fluorescent quenching and lipophilic membrane probes: R18 and FM 1-43. Viability, morphological integrity and functionality of extracted amebocytes appeared to be retained in vitro. The phagocytic properties of L. polyphemus amebocytes described here, in the absence of endotoxin, are similar to those observed for arthropod immunocytes and mammalian neutrophils.

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► Phagocytosis of microbial and synthetic targets by Limulus amebocytes in vitro.
► Optimised conditions for in vitro of maintenance of L. polyphemus amebocytes.
► Amebocyte phagocytic indices are comparable to other invertebrate immune cells.
► Using membrane probes & quenching of fluorescence confirms target internalisation.