Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream ( Sparus aurata L.) by flow cytometry and electron microscopy

Abstract

In this paper we optimize a flow cytometric method for evaluating the phagocytic activity of leucocytes in gilthead seabream (Sparus aurata L.) and characterize the phagocytic cells observed. Optimal conditions were established for the fluorescein-labelling and analysis of the bacterium Vibrio anguillarum by flow cytometry. Head-kidney leucocytes were incubated with the heat-killed fluorescein isothiocyanate (FITC)-labelled bacteria for different periods, during which the kinetics of phagocytosis was studied. Attached and interiorized bacteria were distinguished. Although phagocytic ability reached a maximum after 60 min, phagocytic capacity reached its maximum at 20 min. The amount of ingested bacteria per phagocyte was estimated from the mean fluorescence of the leucocytes. Cytochalasin B or colchicine was used to inhibit phagocytosis. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity as demonstrated by transmission electron microscopy. In conclusion, the technique presented allows the screening of thousands of cells, and individual cell evaluation, by quantifying interiorized particles in fish phagocytes. Our ultrastructural results demonstrate that V. anguillarum is actively phagocytized by seabream macrophages and acidophilic granulocytes.

Superparamagnetic Iron Oxide-Labeled Schwann Cells and Olfactory Ensheathing Cells Can Be TracedIn Vivoby Magnetic Resonance Imaging and Retain Functional Properties after Transplantation into the CNS

“…Evaluation of uptake process for SPIO in vitro. SCs were cultured in multiwell plates and preincubated with either colchicine (Sigma), to inhibit pinocytosis (5, 50, and 500 g/ml), or cytochalasin B (Sigma), to inhibit phagocytosis (10 and 100 g/ml) for 2 hr (Band et al, 1986;Pratten and Lloyd, 1986;Esteban et al, 1998). After preincubation, the cultures were washed with PBS and incubated with SPIO at a concentration equivalent to 2 mg/ml Fe for 24 hr.…”

Glutathione loaded solid lipid nanoparticles: Preparation and in vitro evaluation as delivery systems of the antioxidant peptide to immunocompetent fish cells

“…HK samples were cut into small fragments and transferred to 8 ml of sRPMI [RPMI-1640 culture medium supplemented with 0.35% sodium chloride (to adjust the medium's osmolarity to seabream plasma osmolarity of 353.33 mOs), 2% FCS, 100 I.U. ml −1 penicillin and 100 g/ml streptomycin] for leucocyte isolation [21]. Cell suspensions were obtained by forcing fragments of the organ through a nylon mesh (mesh size 100 m), washed twice (400 g 10 min), counted (Z2 Coulter Particle Counter) and adjusted to 2x10 7 cells/ml in sRPMI.…”

Immunocytochemical Tools Reveal a New Research Field Between the Boundaries of Immunology and Reproductive Biology in Teleosts

“…The use of the G7 antibody has also allowed enriching head-kidney (hkG7+ cells) and testis (tG7+ cells) cell suspensions by magnetic activated cell sorting (MACS). Those enriched cell suspensions together with total testicular and head-kidney cell suspensions have been used in flow cytometry assays to asses phagocytosis (Esteban et al, 1998), reactive oxygen intermediates (ROI) production (Banati et al, 1994) and metalloproteinases activity (Leber and Balkwill, 1997). Combining those techniques, it has been demonstrated that headkidney acidophilic granulocytes infiltrate the gonad in response to gonadal and hormonal stimulus and showed completely different functional capabilities than their head-kidney counterparts (Chaves-Pozo et al, 2005b, 2008a: impaired phagocytic and ROI production activities although they were the majority of cells which were able to produce ROIs in the testis upon phorbol myristate acetate (PMA) stimulation (Chaves-Pozo et al, 2005b).…”

“…In that sense we have first performed the phagocytic assay (review in Esteban et al, 1998), and then the immunostaining keeping the cells at 4ºC to avoid digestion of the phagocyted-fluorescence-bacteria and internalization of the antigen-antibody complex at the membrane. Thus, we have observed that the 95% of the head-kidney acidophilic granulocytes had phagocytic capability in contrast with the 1% of the testicular acidophilic granulocytes (Chaves-Pozo et al, 2005b;Sepulcre et al, 2002).…”