Vibrio harveyi, and Streptococcus iniae were obtained from the National Center for Mariculture (NCM) pathology department from the bacterial stocks kept at − 80 °C. V. harveyi, was originally isolated from spleen of Sparus aurata in 2012 and S. iniae was originally isolated from the liver of a Siganus rivulatus in 2010. Both bacteria were sent for 16S rRNA sequencing at Hy Laboratories Ltd., and were identified, compared, and aligned with those of other V. harveyi, and S. iniae available in the GenBank database (NCBI/BLAST) before further use. Bacteria from the -80 °C were then defrosted to room temperature and inoculated in a laminar flow hood on tryptic soy agar (TSA, DIFCO USA) prepared with 25% sterile seawater, and incubated at 24 ± 1 °C for 48–72 h. After the incubation period, the bacterial isolates were transferred to tryptic soy broth (TSB, ACUMEDIA USA) prepared with 25% sterile seawater and incubated again for another 48–72 h at 24 ± 1 °C. OD values from the bacterial concentration were read at 600 nm using a microplate spectrophotometer (PowerWaveTMXS, BioTek, Winooski, USA).
Powerwavetmxs
Manufactured by Agilent Technologies
Sourced in United States
The PowerWaveTMXS is a high-performance spectrum analyzer designed for a variety of applications. It provides accurate and reliable measurements across a wide frequency range. The core function of the PowerWaveTMXS is to analyze and measure electrical signals, enabling users to identify and characterize various frequency components.
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Lab products found in correlation
Un 500 universal oven, by Memmert (2 mentions)
Lyobeta 15 lyophilizer, by Telstar (2 mentions)
Fp 6200, by Jasco (2 mentions)
Tryptic soy broth (tsb), by Acumedia (1 mentions)
Standard cell culture flasks, by Greiner (1 mentions)
Cellstar multiwell culture plates, by Greiner (1 mentions)
Spss version 22, by IBM (1 mentions)
Agilent triple quadrupole ms detector, by Agilent Technologies (1 mentions)
Agilent 1200 liquid chromatograph, by Agilent Technologies (1 mentions)
Stomacher 400 circulator, by Seward Medical (1 mentions)
13 protocols using powerwavetmxs
1
Isolation and Identification of Marine Bacteria
2
Multimode Microplate Reader Assays
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BioTek powerWaveTM XS (BioTek Instruments, (Winooski, VT, USA), FP-6200 (JASCO, Easton, MD, USA) and Infinite® 200 PRO multimode (TECAN, Deutschland GmbH, Crailsheim, Germany) microplates readers, convection oven (Romag S.A, Barcelona, Spain), UN 500 universal oven (Memmert, Schwabach, Germany) and Telstar Lyobeta-15 lyophilizer (Telstar, Madrid, Spain) were used for analyses. Forcell culture assays, a Neubauer cell counting chamber (0.100 mm depth, 0.0025 mm2) (Paul Marienfeld GmbH & Co., KG, Lauda-Königshofe, Germany), an incubator (BINDER GmbH, Tuttlingen, Germany), a Thermo Scientific™ MSC-Advantage™ class II biological safety cabinet (Thermo Fisher Scientific, Waltham, MA, USA), an autoclave (3870EA, Tuttnauer, Hauppauge, NY, USA), a TR400-SW TRINO microscope (VWR, Vienna, Austria), CELLSTAR® multiwell culture plates and standard cell culture flasks (Greiner Bio-One GmbH, Kremsmuenster, Austria) were used.
3
Viability of Differentiated Neuronal Cells
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The viability of cells was determined by MTT assay. SH-SY5Y cells were plated in 96-well plates (2 × 104 cells/well), cultures for 24 h at 37 °C in 5% CO2 and were differentiated for 5 days with 10 µM RA. The differentiated neurons were treated with serially diluted concentration of catechin (cat), epicatechin (epi), cyanidin (cy) (50–0.1 µM in EtOH/DMSO (1:5 v/v) and red fruit extract (final concentration: 1, 0.5, 0.25, 0.125, 0.62 and 0 mg/mL) dissolved in serum-free DMEM. After 3 and 18 h of incubation 20 µL of a MTT solution (5 mg/mL in PBS) was added to each well and incubated for an additional 2 h at 37 °C in 5% CO2. Formazan crystals formed in the wells were solubilized in 200 µL of DMSO (Dimethyl sulfoxide). Absorbance was measured at 570 nm wavelength employing a microplate reader PowerWaveTM XS (BioTek Instruments, Inc., Winooski, VT, USA).
The assay was repeated with three independent experiments. The viability was calculated in comparison to control experiments in which a solvent control was added in place of polyphenols and that was used as 100% viable reference [53 (link)].
The assay was repeated with three independent experiments. The viability was calculated in comparison to control experiments in which a solvent control was added in place of polyphenols and that was used as 100% viable reference [53 (link)].
4
Isolation and Characterization of Vibrio harveyi
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Vibrio harveyi was isolated from the brain of a flathead grey mullet (Mugil cephalus) presenting spiral swimming behavior during an outbreak in 2016, in a laminar flow hood, and streaked onto tryptic soy agar (TSA, DIFCO), prepared with 25% sterile seawater (40 ppt), and incubated at 24 ± 1 °C for 48 h. After incubation, the bacterium was inoculated in tryptic soy broth (TSB, DIFCO), prepared with 25% sterile seawater (40 ppt), and incubated for another 24 h at 24 ± 1 °C. Bacterial density was measured at OD600 using a microplate spectrophotometer (PowerWave TMXS, BioTek, Winooski, VT, USA). One part of the bacterial suspension was stored at −80 °C in glycerol (1:1) to keep it in stock for future studies.
5
MTT Assay for Cell Viability
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Cell viability was measured by MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay, as described previously [42 (link)] with a slight modification. Briefly, the cells were plated at a density of 1 × 105 cells/well in 24 well plates and cultured overnight in complete DMEM media containing 10% FBS. The cells were then treated with various concentrations (0–300 μg/mL) of RDL and incubated for 24, 48 and 72 hr in serum-free medium. MTT dye (5 mg/mL) was added to the cell culture media and they were incubated for an additional 3 hr. After the medium was removed, DMSO was added to the cells for the solubilization of generated formazan salts. The amount of formazan salt formation was decided by measuring the optical density (OD) at 540 nm using GENios® microplate spectrophotometer (PowerWaveTMXS, BioTek Instruments, Inc., Winooski, VT, USA). Relative cell viability of treatment was calculated as a percentage of vehicle-treated control; OD of treated cells/OD of control × 100.
6
Anti-inflammatory Activity of RDL in P. acnes-Stimulated HaCaT Cells
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The anti-inflammatory activity of RDL was examined in P. acnes-stimulated HaCaT cells. To prepare, the P. acnes culture was harvested and washed with PBS and then centrifuged at 10,000× g for 5 min. After two washes with PBS, the P. acnes pellet was resuspended in DMEM medium without antibiotics. After 30 min, the cells were treated with live P. acnes (wet weight 200 μg/mL) alone or in combination with various concentrations of RDL for 24 h incubation period. Then, cell-free supernatants were collected and the concentrations of TNF-α, IL-8 and IL-1β were determined using a commercially available ELISA kit, according to the manufacturer’s instructions. Reading of the absorbance at 450 nm was performed by GENios® microplate spectrophotometer (PowerWaveTMXS, BioTek Instruments, Inc., Winooski, VT, USA).
7
MTT Assay for Keratinocyte Viability
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Cell viability was measured by MTT assay. Briefly, human keratinocyte cells (HaCaT) were maintained in DMEM with 10% FBS and 100 μg/mL penicillin-streptomycin at 37 °C in a 5% CO2 humidified incubator. HaCaT cells were plated at a density of 4 × 104 cells/well in 24-well plates and cultured overnight in growth DMEM media. Cells were removed by gentle washing with fresh culture medium and treated with various concentrations of RJ and incubation for 24, 48 and 72 h. MTT solution (5 mg/mL) was then added to each well and incubated for an additional 3 h at 37 °C. Finally, DMSO was added to solubilize the formazan salt and the amount of formazan generated was determined by measuring the optical density (OD) at 540 nm using a GENios® microplate spectrophotometer (PowerWaveTMXS, BioTek Instruments, Inc., Winooski, VT, USA).
8
Bacterial Growth Kinetics Determination
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Overnight cultures of all bacterial isolates (P0, P10, and X10) were adjusted to an OD600 of 0.8, further diluted 1:100 in Mueller Hinton broth and deposited into 96 well plates. To determine the planktonic growth rate of bacteria, the culture plate was placed into a microplate reader (PowerWaveTM XS, BioTek, Swindon, United Kingdom) and the optical density was read every hour for 24 h using Gen5TM 1.08 software (BioTek, Swindon, United Kingdom). Growth curve data from eight absorbance readings (biological duplicates each comprising 4 technical replicates) were fitted to a standard form of the logistic equation using the R software package Growthcurver (Sprouffske and Wagner, 2016 (link)) to determine metrics relating to intrinsic growth rates (r; h–1), carrying capacity (K) and maximum generation time (t_gen; h–1). Pairwise statistical comparisons of generated datasets were performed between parent and passaged mutants (P0 vs. P10; P0 vs. X10) at P ≤ 0.05 using a Wilcoxon signed-rank test. Comparisons were performed using SPSS version 22 (IBM analytics, New York, United States).
9
Biofilm Formation Assay of L. plantarum
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Biofilm formation capability of the non-aggregating strain NZ3400 and its aggregating derivative strain PA4_02 was assessed in MRS, minimal medium containing glucose, sucrose, fructose or maltose as sole carbon source and EMS medium as described previously with minor modifications [75 (link)]. Wells of 96-well tissue culture test plates (Bioswisstec AG, Schaffhausen, Switzerland) containing 200 μl of corresponding growth medium were inoculated with 106 CFU/ml L. plantarum. Wells only containing the medium served as control. The plates were incubated at 37 °C for 24 h. The liquid was removed, and the wells were washed three times with phosphate-buffered saline (PBS), pH 6.2. After drying, the wells were supplemented with crystal violet (0.1%, w/v) and incubated for 30 min. The stained biofilm was washed three times with PBS, the dye was resolved in ethanol (99%) and absorbance was measured at 595 nm (PowerWaveTMXS; Bio-Tek Instrument Inc., Winooski, VT, USA).
10
Epicatechin and Cocoa Extract Viability
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The viability of cells was determined by MTT assay. SH-SY5Y cells were plated in 96-well plates (2 × 104 cells/well), cultured for 24 h at 37 °C in 5% CO2 and were differentiated for 5 days with 10 µM RA. The differentiated neurons were treated with serially diluted concentration of epicatechin (epi) or cocoa extract. After 3 and 18 h of incubation, 20 µL of a MTT solution (5 mg/mL in PBS) was added to each well and incubated for an additional 2 h at 37 °C in 5% CO2. Formazan crystals formed in the wells were solubilized in 200 µL of Dimethyl sulfoxide (DMSO). Absorbance was measured at 570 nm wavelength employing a microplate reader PowerWaveTM XS (BioTek Instruments, Inc., Winooski, VT, USA). The viability was calculated in comparison to control experiments in which a solvent control was added in place of epicatechin or cocoa extract and that was used as a 100% viable reference [34 (link)].
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