title: Isolation of mast cells from the peritoneal exudate of the teleost fish gilthead sea bream (Sparus aurata L.).
abstract: Inflammation is the first response of animals to infection or tissue damage. Sparus aurata (Perciformes) was the first fish species shown to possess histamine-containing mast cells at mucosal tissues. We report a separation protocol for obtaining highly enriched (over 95% purity) preparations of fish mast cells in high numbers (5-20 million mast cells per fish). The peritoneal exudate of S. aurata is composed of lymphocytes, acidophilic granulocytes, macrophages and mast cells. We separated the lymphocyte fraction through discontinuous density gradient centrifugation. The remaining cells were cultivated overnight in RPMI-1640 culture medium containing 5% fetal calf serum, which allowed macrophages to adhere to the cell culture flasks. Finally, acidophilic granulocytes were separated from the mast cells though a Magnetic-Activated Cell Separation (MACS) protocol, using a monoclonal antibody against these cells. The purity of mast cells-enriched fractions was analyzed by flow cytometry and by transmission electron microscopy. The functionality of purified mast cells was confirmed by the detection of histamine release by ELISA after stimulation with compound 48/80 and the induction of the pro-inflammatory cytokines IL-1β and IL-8 following stimulation with bacterial DNA. This fish mast cells separation protocol is a stepping stone for further studies addressing the evolution of vertebrate inflammatory mechanisms.
authors.affiliation: Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, IMIB-Arrixaca, Campus Universitario de Espinardo, 30100 Murcia, Spain.
authors.forename: Nuria Esther
authors.initials: NE
authors.lastname: Gómez-González
authors.affiliation: Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, IMIB-Arrixaca, Campus Universitario de Espinardo, 30100 Murcia, Spain.
authors.forename: Erick
authors.initials: E
authors.lastname: García-García
authors.affiliation: Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, IMIB-Arrixaca, Campus Universitario de Espinardo, 30100 Murcia, Spain.
authors.forename: Jana
authors.initials: J
authors.lastname: Montero
authors.affiliation: Instituto Español de Oceanografía, Planta de Cultivos Marinos, Ctra. de la Azohía s/n, 30860 Puerto de Mazarrón, Murcia, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: García-Alcázar
authors.affiliation: Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, IMIB-Arrixaca, Campus Universitario de Espinardo, 30100 Murcia, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Meseguer
authors.affiliation: Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, IMIB-Arrixaca, Campus Universitario de Espinardo, 30100 Murcia, Spain.
authors.forename: Alfonsa
authors.initials: A
authors.lastname: García-Ayala
authors.affiliation: Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, IMIB-Arrixaca, Campus Universitario de Espinardo, 30100 Murcia, Spain. Electronic address: vmulero@um.es.
authors.forename: Victoriano
authors.initials: V
authors.lastname: Mulero
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000662:veterinary*; D005122:Exudates and Transudates / Q000166:cytology; D008407:Mast Cells / Q000166:cytology*; D010537:Peritoneum / Q000166:cytology / Q000378:metabolism; D021541:Sea Bream / Q000502:physiology*
keywords: Evolution; Histamine; Inflammation; Innate immunity; Teleosts