----------------- INFORMACIÓN GENERAL ------------------- 1. Título del dataset Erythrocyte phagocytosis in gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax). 2. Autoría: [Rellenar la información de todos los autores siguiendo el siguiente formato. Repetir el esquema, uno para cada autor.] Nombre: Jose Carlos Campos-Sánchez Institución: Universidad de Murcia Correo electrónico: josecarlos.campos@um.es ORCID: 0000-0003-0303-5412 Nombre: Francisco A. Guardiola Institución: Universidad de Murcia Correo electrónico: faguardiola@um.es ORCID: 0000-0002-1018-5446 Nombre: José Meseguer Institución: Universidad de Murcia Correo electrónico: meseguer@um.es ORCID: - Nombre: María Ángeles Esteban Institución: Universidad de Murcia Correo electrónico: aesteban@um.es ORCID: 0000-0002-6264-1458 3. Fecha de recogida de los datos (fecha única o rango de fechas): [01-07-2022---04-11-2024] 4. Fecha de depósito de los datos: [04-11-2024] 5. Idioma del conjunto de datos: Inglés ------------------------ INFORMACIÓN METODOLÓGICA ------------------------ 1.Descripción de la metodología utilizada para generar el conjunto de datos. 2. Material and methods 2.1. Animals Specimens of the seawater teleost gilthead seabream (S. aurata) (33.0 g ± 2.9 g, 12.4 cm ± 0.3 cm) and European sea bass (D. labrax) (12.6 g ± 1.2 g, 10.9 cm ± 0.3 cm), obtained from a local farm (Murcia, Spain), were kept in re-circulating seawater aquaria (450 L) at the Marine Fish Facilities at the University of Murcia (Spain) during a quarantine period of one month. Water temperature was maintained at 20 ± 2 °C with a flow rate of 900 L h−1, a salinity of 28 ‰, a photoperiod of 12 h light to 12 h dark and with continuous aeration. Water ammonium and nitrite levels in the tanks were maintained below the limits for the species (0.1 mg L−1 and 0.2 mg L−1, respectively). Fish were fed with a commercial diet (Skretting, Spain) at a rate of 2% body weight day−1 and were kept 24 h without feeding before the trial. Fish maintenance, experimental procedures and euthanasia were carried out in compliance with ethical standards, and the protocol was approved by the Ethical Committee for Animal Experimentation of the University of Murcia (permit number A13150104) adhering to the European directive 2010/63/EU on the protection of animals used for scientific purposes. 2.2. Erythrocytes isolation Six specimens of each species were randomly selected and subsequently anesthetized with clove oil (20 mg L-1, Guinama®). Immediately, 0.2 mL of blood was extracted into a heparinized syringe from the caudal vein and transferred into 7 mL of phosphate buffered saline (PBS, Fisher Bioreagents), containing 0.35% sodium chloride to adjust the medium osmolarity, and 10 mM glucose (henceforth referred to as PBS-glu). The fish were then returned to the aquaria in accordance with the 3 Rs principle [39]. The blood was layered over a 51% Percoll density gradient (Pharmacia) and centrifuged (400 x g for 30 min, 4 °C) to separate leucocytes and erythrocytes. The latter cells, located in the pellets, were collected, washed twice with PBS-glu, counted using a TC20 automated cell counter (Bio-Rad), and adjusted to a concentration of 107 cells mL-1 in PBS-glu. 2.3. Phagocytosis assay The phagocytic activity of gilthead seabream and European sea bass erythrocytes was investigated utilizing flow cytometry [40]. Vibrio alginolyticus (strain CECT 586) was cultivated from 1 mL of stock culture previously preserved at −80 °C. Before use, an aliquot of stock culture was incubated with tryptic soy broth (TSB, Difco Laboratories) medium supplemented with 1.5 % NaCl in flasks filled to 10 % capacity. The flasks were incubated at 25 ºC with continuous agitation (100 rpm) until mid-logarithmic growth phase was achieved. The bacterial cells were subsequently harvested by centrifugation at 1,100 x g for 8 min, washed twice with PBS buffer, and fixed in 3 % formalin for 30 min, followed by washing with 0.1 mol·L-1 NaHCO3 [41]. The formalin-inactivated bacteria were then incubated in 0.1 mol·L-1 NaHCO3 containing 0.1 mg·mL-1 fluorescein isothiocyanate (FITC, Sigma) in darkness under gentle rotation at 25 ºC for 3 h. The FITC conjugated bacteria were washed three times and re-suspended in PBS buffer at a final concentration of 108 cells·mL-1. FITC-labelled bacteria were used in the flow cytometric study. The staining uniformity was examined, after which the bacterial suspensions were aliquoted and stored at -80 °C. Heat killed (30 min, 60 °C) lyophilized Saccharomyces cerevisiae (strain S288C) was washed twice, counted, and adjusted to 108 yeasts mL-1 in PBS. To label yeasts with FITC they were incubated with 5 mg mL-1 FITC at 22 °C with constant stirring (40 cycles min-1), in darkness for 15 min [42]. After labelling, free FITC was removed by washing twice in PBS and the yeasts were resuspended in PBS. FITC-labelled yeasts were obtained for flow cytometric analysis. The uniformity of staining was evaluated, and subsequently, the yeast suspensions were aliquoted and stored at -80 °C. Phagocytosis samples consisted of 100 μL (106 cells mL-1) of erythrocytes and 50 μL of V. alginolyticus (5 x 107 cells mL-1) or 50 μL of S. cerevisiae (5 x 107 cells mL-1) to obtain a ratio of cells to bacteria or yeasts of 1-50. These samples were mixed, centrifuged (400 x g, 5 min, 22 °C) and incubated at 22 °C for 20, 40, 60, 80, 100 or 120 min. Afterwards, the samples were placed on ice to stop phagocytosis and diluted by 400 μL ice-cold PBS. The fluorescence of the extracellular bacteria or yeasts was quenched by adding 50 μL ice-cold trypan blue (0.4 % in PBS). Standard samples of FITC-labelled V. alginolyticus, FITC-labelled S. cerevisiae or erythrocytes were incorporated in each phagocytosis assay. All samples were analysed in a flow cytometer (Becton Dickinson) with an argon-ion laser adjusted to 488 nm. Analyses were performed on 5,000 cells, which were acquired at a rate of 300 cells s−1. Data were collected in the form of two-parameter side scatter (granularity) (SSC) and forward scatter (size) (FSC), and green fluorescence (FL1) dot plots or histograms were made on a computerized system. The fluorescence histograms represented the relative fluorescence on a logarithmic scale. The cytometer was set to analyse the phagocytic cells showing the highest SSC and FSC values. Phagocytic ability was defined as the percentage of cells with one or more ingested bacteria or yeast (green-FITC fluorescent cells) within the erythrocyte cell population whilst the phagocytic capacity was the relative number of ingested bacteria or yeasts per erythrocyte, and it was assessed in arbitrary units from the mean fluorescence intensity of the phagocytic cells. 2.4. Respiratory burst activity The respiratory burst activity of erythrocytes was investigated utilizing a chemiluminescence method [43]. Briefly, 100 μL of erythrocytes, previously incubated with the suspension of bacteria or yeast for 20, 40, 60, 80, 100 or 120 min, were placed in a 96-well flat-bottomed plate. Subsequently, 100 μL of Hank’s balanced salt solution (HBSS, Gibco) containing 1 μg mL-1 phorbol myristate acetate (PMA, Sigma) and 10-4 M luminol (Sigma) was added to each well. The plate was agitated and immediately analysed in a chemiluminometer (BMG, FluoStar Omega) for 30 min at 2-min intervals. The kinetics of the reactions were analysed every 2 min for 30 min, and the slope min-1 of each curve was calculated. Luminescence backgrounds were calculated using reagent solutions containing luminol but not PMA. 2.5. Light and transmission electron microscopies Pellets from the phagocytic assay were directly placed on a slide and observed under a fluorescent light microscope (Axioskop Zeiss). Conversely, erythrocytes incubated with the bacteria or yeast suspension as previously described were centrifuged (400 x g, 5 min, 22 °C), washed in 250 μL of PBS-glu and immediately fixed with 200 μL of 2.5 % glutaraldehyde in 0.1 M cacocylate buffer, pH 7.2–7.4 for 5-10 min at 4°C [44]. For transmission electron microscope (TEM), samples were then post-fixed for 2 h in 1 % OsO4 and embedded in Epon. Semithin and ultrathin sections were obtained with a Reichert-Jung Ultracut and stained with toluidine blue and contrasted with uranyl acetate and lead citrate, respectively. Ultrathin sections were examined with a Zeiss EM 10C electron microscope. 2.6. Statistical analysis The results were expressed as mean ± standard error of the mean (SEM). Data from the phagocytic assay were analysed by one-way ANOVA (followed by Tukey tests) to determine differences between the various experimental time points assessed. Normality of the data was previously evaluated using a Shapiro-Wilk test, and homogeneity of variance was verified using the Levene test. All statistical analyses were conducted using the SPSS software package (version 28.0; SPSS Inc., Chicago, IL, USA) for Windows. The level of significance was set at p < 0.05 for all statistical tests. 2. Software o instrumentos necesarios para interpretar los datos: [Incluir la versión del software. Si hace falta un software específico de acceso restringido, explicar cómo obtenerlo. Valorar si es posible cambiar el conjunto de datos a un formato abierto (recomendado).] Data analysis: Computer package SPSS (25.0 version; SPSS Inc., Chicago, IL, USA) for Windows. 3. Procedimientos seguidos para asegurar la calidad de los datos Positive and negative controls in each assay. Samples and controls in triplicates to avoid pipetting errors. ------------------------ ESCTRUCTURA DE LOS ARCHIVOS --------------------------- 1. Nombres de archivos [Mencionar todos los archivos incluidos en el conjunto de datos, con el nombre y la extensión (.csv, .pdf, etc.) de cada fichero]. Raw data.xls 2. Formato de los archivos: Libro de Excel 97-2003 (*.xls) ------------------------ MÁS INFORMACIÓN ------------------------ [Incluir cualquier otra información sobre el conjunto de datos que no haya quedado reflejada en esta plantilla y que se considere relevante.]