----------------- INFORMACIÓN GENERAL ------------------- 1. Título del dataset In vitro immune-depression and anti-inflammatory activities of cantharidin on gilthead seabream (Sparus aurata) leucocytes activated by λ-carrageenan 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: 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): [13-05-2021---01-10-2021] 4. Fecha de depósito de los datos: [21-11-2023] 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. Fish Sixty specimens (323.90 g ± 13.81 g, 26.42 cm ± 0.43 cm) of the seawater teleost gilthead seabream (S. aurata) were obtained from a local farm (Murcia, Spain), randomly redistributed in ten tanks (450 L) and kept in re-circulating seawater at the Marine Fish Facilities at the University of Murcia (Spain) during a quarantine period of one month. Water temperature was kept at 20 ± 2 °C with a flow rate of 900 L h−1, the salinity was of 28 ‰, a photoperiod artificial of 12 h light to 12 h dark was established and with continuous aeration. Water ammonium and nitrite levels in the tanks were maintained below 0.1 mg L−1 and 0.2 mg L−1, respectively. Fish were fed a commercial diet (Skretting, Spain) at a rate of 2 % body weight day−1 and were kept 24 h without feeding before sampling. All experimental protocols were approved by the Ethical Committee of the University of Murcia. 2.2. Head-kidney leucocytes isolation Six fish were randomly selected and anesthetized with clove oil (at a concentration of 20 mg L-1, Guinama®). After anaesthesia, they were bleeding and the head kidney was removed through a ventral incision, cut into small pieces, and placed in 12 mL of sRPMI. This sRPMI consists of RPMI-1640 culture medium (Gibco) supplemented with 0.35% sodium chloride (to match the osmolarity of the medium to the osmolarity of seabream plasma, which is 353. 33 mOs), 3% foetal calf serum (FCS, from Gibco), as well as 100 international units (i.u). mL-1 of penicillin and 100 mg mL-1 of streptomycin. (Life Technologies)] [24]. HKLs suspensions were obtained by forcing fragments of the organ through a nylon mesh (mesh size 100 μm), washed twice (400 x g 10 min), counted (Z2 Coulter Particle Counter) and adjusted to 2 x 107 cells mL-1 in sRPMI. Cell viability, assessed by trypan blue exclusion test with a Bio-Rad TC20 automated cell counter, exceeded 98%. 2.3. Leucocyte incubation Cantharidin (Sigma) was diluted into dimethyl sulfoxide (DMSO, Sigma) and then resuspended in sRPMI to obtain final dilutions. λ-Carrageenan (Sigma) was diluted into sterile PBS and a stock solution of 10 mg mL-1 was prepared. Then, the stock solution of λ-carrageenan was resuspended in sRPMI and serial dilutions for each concentration were prepared. Pryor to carrying out the assays, the osmolarity of these solutions was measured in an osmometer (Wescor) to avoid effects due to this parameter. For each fish and assay, samples of 500 μL of seabream HKLs (previously adjusted to 2 x 107 cells mL-1) were dispensed into 24-well plates (Nunc). HKLs were incubated with 250 μL of cantharidin [at a final concentration of 0 (DMSO diluted in sRPMI; control), 2.5 and 5 μg mL-1] and 250 μL of λ-carrageenan at a final concentration of 0 (PBS diluted in sRPMI; control). Cells were incubated for 24 h at 25 ºC, 85 % humidity, and 5 % CO2. Each assay was done with HKLs from 6 specimens (not pooled HKLs). 2.4. Leucocyte viability In order to determine the gilthead seabream HKLs viability, the number of viable leucocytes were evaluated by a flow cytometry technique using propidium iodide (PI), a membrane impermeant dye that is generally excluded from viable cells [25]. Briefly, 50 μL of PI (400 mg mL-1, red fluorescence, Sigma) was added to each 100 μL aliquot of HKLs (previously incubated with the different mixtures elaborated from cantharidin and λ-carrageenan as described above). The samples were gently mixed and incubated (5 min, 22 °C) before analysis in a FACScan (Becton Dickinson, Madrid, Spain) flow cytometer with an argon-ion laser adjusted to 488 nm. Analyses were performed on 10,000 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 red fluorescence (FL2). Dot plots or histograms were made on a computerized system. Positive controls consisted of HKLs lysed 50 μL with 0.02% cetyltrimethyl ammonium bromide (CTAB, Sigma), shaken (10 min, 60 rpm, in the dark), and mixed with 50 μL of PI. Dead cells were estimated as the percentage of cells with PI (red-PI fluorescent cells). For each sample, total HKL viability was determined, as well as specific viability for three types of leukocytes; for the latter, three regions [R1: acidophilic granulocytes (AGs), R2: monocytes/macrophages (MMs), and R3: lymphocytes] were established in dot plots according to their SSC and FSC [20]. 2.5. HKLs immune activities To determine the peroxidase activity in HKLs, aliquots of 5 μL of HKLs (previously incubated with the mixtures of cantharidin and λ-carrageenan) in sRPMI were placed in triplicate wells of a 96-well flat-bottomed plate. The cells were lysed with 50 μL of 0.02% CTAB (Sigma) and shaken for 10 min at 60 RPM in the dark. Afterwards, 100 μL of 20 mM 3, 3´, 5, 5´ - tetramethyl benzidine hydrochloride (TMB, Sigma) and 5 mM H2O2 was added per well. The colour change reaction was stopped after 2 min by adding 50 μL of 2M H2SO4, and the optical density was read at 450 nm in a plate reader (BMG, FluoStar Galaxy) [26]. Standard samples without leucocytes were used as blanks. The respiratory burst activity of HKLs was studied by a chemiluminescence method [27]. Briefly, 100 μL of HKLs, previously incubated, were placed in triplicate wells of a 96-well flat-bottomed plate. Then, 100 μL of HBSS (Hank’s balanced salt solution, 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 shaken and immediately read in a chemiluminometer (BMG, FluoStar Omega) for 30 min at 2-min intervals. The kinetic of the reactions was analysed every 2 min during 30 min, and the slope min-1 of each curve was calculated. Luminescence backgrounds were calculated using reagent solutions containing luminol but not PMA. The phagocytic activity of gilthead seabream HKLs was studied by flow cytometry [28]. Heat killed (30 min, 60 °C) lyophilized Saccharomyces cerevisiae, strain S288C, were washed twice, counted, and adjusted to 1 x 108 yeast cells mL−1 in RPMI. To label yeast cells with fluorescein isothiocyanate (FITC, Sigma) they were incubated with 5 mg mL-1 FITC at 22 °C with constant stirring (40 cycles min−1) and in darkness for 15 min [29]. After labelling, free FITC was removed by washing twice in PBS and the yeast cells were resuspended in RPMI. FITC-labelled yeast cells were acquired for flow cytometric study. The staining uniformity was examined and then the yeast cell suspensions were aliquoted and stored at -80 °C. Phagocytosis samples consisted of 100 μL of HKLs (incubated with the different mixtures elaborated from cantharidin and λ-carrageenan) and 60 μL of S. cerevisiae (5 x 107 cells mL-1 of RPMI) which were mixed and centrifuged (400 x g, 5 min, 22 °C), before being resuspended and incubated at 22 °C for 30 min. Then, samples were placed on ice to stop phagocytosis and diluted by 400 μL ice-cold PBS. The fluorescence of the extracellular yeast cells was quenched by adding 40 μL ice-cold trypan blue (0.4% in PBS). Standard samples of FITC-labelled S. cerevisiae or HKLs were included 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 SSC and FSC, and green fluorescence (FL1). 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 yeast (green-FITC fluorescent cells) within the phagocytic cell population, whilst the phagocytic capacity was an approximation to the relative number of ingested yeast cells per cell, and it was assessed in arbitrary units from the mean fluorescence intensity of the phagocytic cells. 2.6. HKLs ultrastructure To determine possible morphological alterations in HKLs after incubation with cantharidin and λ-carrageenan, the samples were processed and studied by transmission electron microscopy (TEM) [30]. After exposure for 24 h with the corresponding mixture, HKLs were centrifuged (400 x g, 5 min, 22 °C), washed in 250 μL of sRPMI and immediately fixed with 200 μL of 2.5% glutaraldehyde in 0.1 M cacocylate buffer (pH 7.2–7.4, 5-10 min, 4°C), then postfixed for 2 h in 1 % OsO4 and embedded in Epon. Semithin and ultrathin sections were obtained with a Reichert-Jung Ultracut, stained with toluidine blue and counterstained with uranyl acetate and lead citrate, respectively. Ultrathin sections were examined with a transmission electron microscope (TEM, Zeiss EM 10C). 2.7. HKLs gene expression by real-time PCR The sequences of the selected genes were obtained from a gilthead seabream database [31]. The Open Reading Frames (ORF) were located using the ExPASy translation software (SIB Bioinformatics Resource Portal) and an additional check was performed using NCBI BLAST sequence alignment analysis (NIH). The primers were designed with the Thermo Fisher OligoPerfectTM tool, according to the following criteria: i) each individual oligonucleotide was composed of 20 nucleotides, ii) the size of the amplicon had between 100 and 120 nucleotides, iii) with a guanidine-cytosine (GC)% between 55 % and 60 %, iv) a semi-naturalization temperature (Melting temperature) as close as possible to 60 °C, and v) the selection of primers that self-inhibit forming hairpins was avoided as far as it was possible, in order not to hinder the amplification reaction. The primer used are presented in Table 1. After 24 h of the incubation of the HKLs with the different combinations of cantharidin and λ-carrageenan, cells were obtained by centrifugation (400 x g, 10 min, 22° C), and the pellets were stored at -80°C for gene expression analysis. Total RNA was extracted with PureLink® RNA Mini Kit (Life Technologies) as indicated by the manufacturer and the quantification and purification was assessed by using the Nanodrop® spectrophotometer; the 260:280 ratios were 1.8–2.0. The RNA was then treated with DNase I (Promega) to remove genomic DNA contamination. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the SuperScript IV reverse transcriptase (Life Technologies) with an oligo-dT18 primer. In the present study, the expression of the selected genes (see Table 1) was evaluated by real-time qPCR with QuantStudio™ Real-Time PCR System Fast (Life Technologies). Reaction mixtures [containing 5 µL of SYBR Green supermix, 2.5 µL of primers (0.6 µM each) and 2.5 µL of cDNA template] were incubated for 10 min at 95 ºC, followed by 40 cycles of 15 s at 95 ºC, 1 min at 60 ºC, and finally 15 s at 95 ºC, 1 min at 60 ºC and 15 s at 95 ºC. The gene expression was analysed using the 2−ΔCt method [32], which was performed as described elsewhere [33]. The specificity of the reactions was analysed using samples without cDNA as negative controls. For each mRNA, gene expression was normalized with the geometric mean of ribosomal protein (s18) and elongation factor 1-alfa (ef1a) RNA content in each sample. Gene names follow the accepted nomenclature for zebrafish (http://zfin.org/). In all cases, each PCR was performed on triplicate samples. 2.8. Statistical analysis The results were expressed as mean ± standard error of the mean (SEM). Data were analysed by Two-way ANOVA (followed by Tukey tests) to determine differences between increasing concentrations doses of cantharidin and λ-carrageenan. Normality of the data was previously assessed using a Shapiro-Wilk test and homogeneity of variance was also verified using the Levene test. All statistical analyses were conducted using the computer package SPSS (25.0 version; SPSS Inc., Chicago, IL, USA) for Windows. The level of significance used was 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]. EA2 - 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.]