----------------- INFORMACIÓN GENERAL ------------------- 1. Título del dataset In vitro effects of a natural marine algae polysaccharide (λ-carrageenin) on seabream erythrocytes, tumour cell lines and marine bacterial pathogens 2. Autoría: Jose Carlos Campos-Sánchez, Francisco A. Guardiola, María Ángeles Esteban* [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): [02-02-2021---01-09-2021] 4. Fecha de depósito de los datos: [08-05-2023] 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. λ-carrageenin The λ-carrageenin (Sigma) was diluted in sterile phosphate-salt buffer (PBS; 11.9 mM phosphate, 137 mM NaCl and 2.7 mM KCl, pH 7.4) (Fisher Bioreagents) and used at concentrations of 0.1-1,000 µg mL-1. Subsequently, λ-carrageenin was resuspended in the appropriate medium depending on the technique used. Thus, λ-carrageenin was resuspended and adjusted to each concentration in PBS with sodium chloride and glucose for incubation with erythrocytes, Eagle's minimal essential medium (EMEM, Thermo Fisher) for HeLa and PLHC-1 cell lines, L-15 Leibowitz medium (Gibco) for SAF-1 cell line, tryptic soy broth (TSB, Difco Laboratories) for V. harveyi, V. anguillarum and P. damselae, or Flexibacter maritimus medium (FMM, Conda) for T. maritimum, and dilutions were prepared for each concentration. Erythrocytes, cell lines or bacteria were exposed to a final solution of 0 (PBS diluted in the same volume of culture medium; control), 0.1, 1, 1, 10, 100 and 1,000 μg mL-1. Before performing the assays, the osmolarity of these solutions was measured in an osmometer (Wescor) to avoid effects due to this parameter. 2.2 Hemagglutinating and hemolytic activity and morphological changes in gilthead seabream erythrocytes Specimens of the sea water gilthead seabream (Sparus aurata L.) (mean weight: 403.6 ± 16.5 g; mean length 27.3 ± 0.3 cm), obtained from a local farm (Murcia, Spain), were kept in recirculating seawater aquaria (450 L) at the Marine Fish Facility of the University of Murcia (Spain) during a quarantine period of one month. The conditions were as follows: water temperature of 20 ± 2ºC, flow rate of 900 L h-1, salinity of 28 ‰, photoperiod of 12 h light to 12 h dark and continuous aeration. Ammonium and nitrite levels in the tank water were maintained below the limits for the species (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 maintained 24 h without feeding prior to the trial. All experimental protocols were approved by the Ethical Committee of the University of Murcia. For erythrocyte isolation, six fish were randomly selected, anesthetized with clove oil (20 mg L-1, Guinama®). Immediately, 1 mL samples of blood were drawn with a heparinized syringe by tail vein injection and placed in 7 mL of PBS, containing 0.35 % sodium chloride, to adjust the osmolarity of the medium, and 10 mM glucose (hereafter, PBS-glu), and the fish were returned to the aqua [24]. The blood was placed on a 51 % Percoll density gradient (Pharmacia) and centrifuged (400 x g, 30 min, 4 °C) to separate leucocytes from erythrocytes. The latter cells, which were in the pellets, were collected, washed twice with PBS-glu, counted and adjusted to 5 x 108 cells mL-1. The hemagglutination properties of λ-carrageenin were carried out by incubating erythrocytes in a 96-well U-shaped plate (Nunc). Briefly, 50 µl of varying concentrations of λ-carrageenin in PBS-glu were placed in each well in sextuplicate and 25 µL of the erythrocyte suspension was added. Erythrocyte hemagglutination was visualized after 1.5, 3 and 6 h of incubation with λ-carrageenin at room temperature, when the erythrocytes in the wells considered as blank had completely sedimented [25]. Positive or negative controls consisted of 25 µL of the erythrocyte suspension and 50 µL of Concanavalin A (lectin that strongly binds erythrocytes) or PBS-glu, respectively. Macroscopic images of the plate were taken from above and below with a Canon 7D camera with a 22 mm 4.5 wide-angle lens (Canon EF) attached to a ring flash with tripod and with a scanner (CanoScan-4400F), respectively. Erythrocyte lysis causes the release of their contents, hemoglobin, either in the form of oxyhemoglobin (HbO2) or deoxyhemoglobin, being the most abundant protein. Thus, erythrocyte viability was determined by hemolysis and subsequent release of oxyhemoglobin to the medium [24]. After 3, 6, 12 and 24 h of exposure of erythrocytes with varying concentrations of λ-carrageenin in PBS-glu, samples were centrifuged (10,000 x g, 1 min) and 100 µL of the supernatant was transferred to 96-well flat-bottom plates (Nunc) and absorbance was measured at 414 nm (the maximum absorbance for oxyhemoglobin) with a plate reader (BMG, Fluoro Star Galaxy). Positive (maximum hemolysis and absorbance) or negative (minimum hemolysis and absorbance) controls consisted of 100 µL of erythrocytes in 1 mL of sterile distilled water or 1 mL of PBS-glu, respectively. Cell morphology was studied by observing the cells with a phase contrast microscope (Zeiss). For this purpose, 500 µL aliquots containing 200,000 cells per well were seeded in 24-well plates with 500 µL per well of 0, 100, and 1,000 μg mL-1 of λ-carrageenin in duplicate, and the cells were incubated for 12 and 24 h at 25°C. 2.3. Cytotoxic activity and morphological changes in cells lines The established cell line PLHC-1 (ATCC® CRL2406™), a hepatocellular carcinoma from Poeciliopsis lucida, was seeded in 25 cm2 plastic tissue culture flasks in EMEM (Thermo Fisher) supplemented with 5% fetal bovine serum (FBS, Life Technologies), 2 mM mL-1 glutamine, 100 i.u. mL-1, penicillin/streptomycin, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate, 95% (Life Technologies). Cells were grown at 30°C in a humidified atmosphere (85 % humidity) and 5% CO2. Exponentially growing cells were detached from the culture flasks by brief exposure to of trypsin (0.05% in PBS, pH 7.2-7.4), according to the standard trypsinization methods. The established SAF-1 cell line (ECACC nº 00122301), fibroblasts from gilthead seabream fin, was seeded in 25 cm2 plastic tissue culture flasks (Nunc) in L-15 Leibowitz medium (Gibco), supplemented with 10% FBS (Life Technologies), 2 mM mL-1 glutamine, 100 i.u. mL−1 penicillin and 100 mg mL−1 streptomycin (Life Technologies). Cells were grown at 25°C in a humidified atmosphere (85 % humidity). Exponentially growing cells were detached from culture flasks by brief exposure to trypsin (0.25 % in PBS, pH 7.2-7.4), according to the standard trypsinization methods. The detached cells were collected by centrifugation (200 x g, 5 min, 25°C) and cell viability was determined as previously described. The established HeLa cell line (ECACC nº 93021013), human cervical cancer cells was seeded in 25 cm2 plastic tissue culture flasks (Nunc) in EMEM (Thermo Fisher) supplemented with 10 % FBS (Life Technologies), 2 mM mL-1 glutamine, 100 i.u. mL-1 penicillin/streptomycin and 0.1 mM non-essential amino acids (Life Technologies). Cells were grown at 37ºC in a humidified atmosphere (85% humidity) and 5% CO2. Exponentially growing cells were detached from the culture flasks by brief exposure to of trypsin (0.05% in PBS, pH 7.2-7.4), as previously indicated. The detached cells from the three cell lines were collected by centrifugation (200 x g, 5 min, 37°C) and the cell viability determined as previously described. Aliquots of 100 µL were dispensed into 96-well tissue culture plates (containing 30,000 cells well-1) and incubated (24 h, at the optimum temperature for each cell line). This cell concentration was previously determined to obtain satisfactory absorbance values in the cytotoxic assay and to avoid cell overgrowth. The culture medium was then replaced with 200 µL per well of the λ-carrageenin concentrations to be tested at the appropriate dilution. Cells were incubated for 3, 6, 12 and 24 h and then viability was determined by the MTT assay, which is based on the reduction of the soluble yellow tetrazolium salt (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide) (MTT, Sigma) to an insoluble blue formazan product by mitochondrial succinate dehydrogenase [26,27]. After incubation with λ-carrageenin dilutions, the medium was removed and 200 µL per well of MTT (1 mg mL-1 in culture medium for each cell line) was added. After 4 h of incubation (at the corresponding temperature for each cell line), the formazan crystals were solubilized with 100 µL per well of dimethyl sulfoxide (DMSO, Sigma). Plates were shaken (5 min, 100 rpm) under dark conditions and absorbance was determined at 570 nm and 690 nm in a microplate reader. Cell morphology was directly observed and photographed from the culture plates using a phase contrast microscope (Zeiss). Each cytotoxicity assay with each cell type was performed in three replicates for each concentration of λ-carrageenin. 2.4. Bactericidal and bacteriostatic activities Four opportunist marine pathogenic bacteria (V. harveyi strain Lg 16/00, V. anguillarum strain CECT4344, P. damselae subsp. piscicida strain PP3 and T. maritimum strain CECT4276) were used in the bactericidal and bacteriostatic assays. All bacterial strains were grown from 1 mL of stock culture that had been previously frozen at −80 °C. Before use, an aliquot of stock culture from V. harveyi, V. anguillarum and P. damselae was incubated with TSB (Difco Laboratories) medium supplemented with 1.5% NaCl, whilst an aliquot of stock culture from T. maritimum was incubated with FMM (Conda) in flasks which 90% empty. The flasks were incubated at 25ºC with continuous shaking (100 rpm) overnight. Exponentially growing bacteria were resuspended in the sterile corresponding culture medium and adjusted to 108 colony-forming units (c.f.u.) mL-1, according to the McFarland standard curve. Samples of 20 µL of λ-carrageenin dilutions previously adjusted in PBS were added (in triplicates) to the wells of a 96-well U-shaped plate (Nunc). Aliquots of 20 µL of the previously cultured bacteria were added to obtain 40 µL final volumes and the plates were incubated for 5 h at 25°C for the bactericidal assay [28]. PBS solution was added to some wells instead of the λ-carrageenin and served as positive control, while culture medium without bacteria was added in other wells to ensure the sterility of the tests. Control samples were incubated under the same experimental conditions as described above. Then, 25 µL of MTT (1 mg mL−1) were added to each well and the plates were newly incubated (10 min, 25ºC) to allow the formation of formazan. Plates were then centrifuged (2,000 x g, 10 min), and the precipitates dissolved in 200 µL of DMSO, of which 100 µL of were transferred to a flat-bottom 96-well plate (Nunc). The absorbance of the dissolved formazan was measured in a spectrophotometer (BMG, SpectroStarnano) at 570 nm and 690 nm in a microplate reader. Bactericidal activity was expressed as percentage of non-viable bacteria, calculated as the difference between absorbance of surviving bacteria compared to the absorbance of bacteria from positive controls (100 %). Aliquots of 100 µL of λ-carrageenin dilutions previously adjusted in PBS were added (in triplicates) to the wells of a flat-bottom 96-well plate (Nunc). Samples of 100 µL of the previously cultured bacteria were added to obtain 200 µL final volumes and the plates were incubated at 25°C. The turbidity of the samples was measured in a spectrophotometer at 620 nm every 120 min for 24 h [29]. PBS solution was added to some wells instead of the λ-carrageenin and served as positive control to evaluate the growth of bacteria without treatment, while culture medium alone was added in other wells to ensure the sterility of the tests. Control samples were incubated under the same experimental conditions as described above. 2.5. Statistical analysis The results were expressed as mean ± standard error of the mean (SEM). Data were analysed by One-way ANOVA (followed by Tukey tests) to determine differences between the doses of λ-carrageenin tested. 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]. EC6 - 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.]