Five percent of each sample filter was cut into small pieces and extracted with distilled water by ultra-sonication. A portion of the extract was filtrated using a syringe filter (DISMIC-25CS, Advantec Co., Ltd., Tokyo, Japan). SO42− and NO3− were measured using an ion chromatograph (600E/700, Waters Co., Ltd., Milford, MA, USA) with an anion suppressor (ASRS 300, Thermo Fisher Scientific, Waltham, MA, USA) and an anion-exchange column (AS4A-SC, Thermo Fisher Scientific, Waltham, MA, USA) [15 ]. The LOQ of SO42− and NO3− were as follows: SO42−, 46 ng/m3; and NO3−, 131 ng/m3.
Dismic 25cs
Manufactured by Advantec
Sourced in Japan
The DISMIC-25CS is a benchtop digital microscope designed for laboratory applications. It features a 2.5-inch high-resolution color LCD display and supports magnification up to 1000x. The microscope is equipped with a USB interface for image capture and data transfer.
Automatically generated - may contain errors
Lab products found in correlation
Model cm 1000, by Eyela (2 mentions)
Triglyceride e test wako, by Fujifilm (2 mentions)
Asrs 300, by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Nissui Pharmaceutical (1 mentions)
717 plus autosampler, by Waters Corporation (1 mentions)
Lc 10ad pump, by Shimadzu (1 mentions)
Spd 10a vp uv vis detector, by Shimadzu (1 mentions)
Cation h guard column, by Bio-Rad (1 mentions)
Cto 10a column oven, by Shimadzu (1 mentions)
Whatman filter paper, by Cytiva (1 mentions)
N hydroxysuccinimide, by GE Healthcare (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Analyst 600, by PerkinElmer (1 mentions)
Agilent 1100 infinity hplc system, by Agilent Technologies (1 mentions)
14 protocols using dismic 25cs
1
Quantifying Anion Levels in Samples
2
Triglyceride Quantification from Microbial Culture
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One milliliter of five-fold diluted culture broth was transferred into a test tube (18×110 mm) containing 2.5 g of glass beads (φ, 1.00-1.05 mm) , and the test tube was shaken at 2,500 rpm for 60 min with a mixer (Model CM-1000; EYELA) . Twenty microliters of the homogenate was mixed with three milliliters of Cleantech TG assay kit reagents (Triglyceride E-test Wako; Wako Pure Chemical Industries, Ltd.) ; this mixture was incubated at 37℃ for 10 min. After centrifugation at 2,500×g for 5 min, the supernatants were filtered using a 0.45-μm membrane filter (DISMIC-25CS; ADVANTEC) . The absorbance of the filtrates was then measured at 600 nm.
3
Porcine Gastric Mucin Preparation
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Porcine gastric mucin (PGM) (mucin type III; Sigma-Aldrich, St Louis, MO) was rehydrated before use in phosphate-buffered saline (PBS) to a concentration of 10% (w/v) by vigorous stirring for 6 hr at 25°C. Insoluble material was removed by centrifugation (16,000 × g, 30 min, 4°C), and the supernatant was dialyzed using a 12-16 kDa molecular weight cut-off membrane at 4°C against PBS for 32 hr with a minimum of six external dialysis solution changes. 24 The dialyzed mucin was diluted to 5% (w/v) in PBS before sterile filtration through a 0.20 μm cellulose acetate membrane syringe filter (DISMIC-25CS; Advantec Toyo, Tokyo, Japan). The sterile PGM was stored at 4°C as a stock solution.
4
Propagation of Diverse Viruses
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MHV A59 strain (VR-764, American Type Culture Collection, Manassas, VA, USA) was propagated on DBT cells. DBT cells were grown in Eagle's minimum essential medium (MEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 5% fetal bovine serum (FBS) in a 75 cm2 flask. Semi-confluent DBT cells were inoculated with MHV and incubated in MEM with 1% FBS at 37 °C (5% CO2) for 3 days. Then, the flask was frozen and thawed once to recover MHV from the cells. The suspensions were centrifuged at 3500g for 15 min. The supernatant was filtered through a 0.2-μm cellulose acetate MEMbrane (DISMIC-25CS, Advantec, Tokyo, Japan).
Bacteriophage phi6 (NBRC 105899, National Institute of Technology and Evaluation (NITE), Tokyo, Japan) was propagated using P. syringae (NBRC14084, NITE) as the host bacterium. To prepare the phi6 stock, P. syringae was propagated in Luria-Bertani broth at 28 °C for 6 h, subsequently inoculated with phi6, and incubated overnight. The suspensions were centrifuged at 3500g for 15 min. The supernatant was filtered through a 0.2-μm cellulose acetate MEMbrane (Advantec).
MNV S7-PP3 strain was propagated on RAW 264.7 cells, as described elsewhere (Kitajima et al., 2008 (link)). All propagated virus stocks were stored at 4 °C before the experiment.
Bacteriophage phi6 (NBRC 105899, National Institute of Technology and Evaluation (NITE), Tokyo, Japan) was propagated using P. syringae (NBRC14084, NITE) as the host bacterium. To prepare the phi6 stock, P. syringae was propagated in Luria-Bertani broth at 28 °C for 6 h, subsequently inoculated with phi6, and incubated overnight. The suspensions were centrifuged at 3500g for 15 min. The supernatant was filtered through a 0.2-μm cellulose acetate MEMbrane (Advantec).
MNV S7-PP3 strain was propagated on RAW 264.7 cells, as described elsewhere (Kitajima et al., 2008 (link)). All propagated virus stocks were stored at 4 °C before the experiment.
5
Quantitative Analysis of Intracellular TAGs
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Cell disruption for analysis of TAGs in intracellular fat globules: A test tube containing 2.5 g of glass beads (ø, 1.00-1.05 mm) and 1 mL of cell suspension with 10 8 -10 9 cells was shaken with a mixer (Model CM-1000; EYELA) at 2,500 rpm until complete cell disruption (approximately 60 min) (Naganuma et al., 1984) .
Quantitative estimation of TAGs: The homogenate (20 mL) was transferred into a test tube containing 3.0 mL of Cleantech TG assay kit reagents (Triglyceride E-test Wako; Wako Pure Chemical Industries, Ltd.). The solution was incubated at 37∞C for 10 min and centrifuged at 2,500 ¥ g for 5 min. The supernatants were filtered using a 0.45-mm membrane filter (DISMIC-25CS; ADVANTEC). The absorbance of the filtrates was then measured at 600 nm (Naganuma et al., 1982) .
Measurement of cell number: The cell number was measured using a counting chamber (Thoma deep 0.1 mm; Erma) (Naganuma et al., 1975) . Calculation of the concentration (quantitative) of TAGs per 10 8 cells: The amount of TAGs accumulated in the intracellular fat globules (TAGs [mg]/10 8 cells) was estimated as follows: (TAGs [mg]/culture medium [mL])/(cell number/culture medium [mL]) ¥ 10 8 . The amount of TAGs in 10 8 cells was defined as the quantitative lipid-accumulation ability.
Quantitative estimation of TAGs: The homogenate (20 mL) was transferred into a test tube containing 3.0 mL of Cleantech TG assay kit reagents (Triglyceride E-test Wako; Wako Pure Chemical Industries, Ltd.). The solution was incubated at 37∞C for 10 min and centrifuged at 2,500 ¥ g for 5 min. The supernatants were filtered using a 0.45-mm membrane filter (DISMIC-25CS; ADVANTEC). The absorbance of the filtrates was then measured at 600 nm (Naganuma et al., 1982) .
Measurement of cell number: The cell number was measured using a counting chamber (Thoma deep 0.1 mm; Erma) (Naganuma et al., 1975) . Calculation of the concentration (quantitative) of TAGs per 10 8 cells: The amount of TAGs accumulated in the intracellular fat globules (TAGs [mg]/10 8 cells) was estimated as follows: (TAGs [mg]/culture medium [mL])/(cell number/culture medium [mL]) ¥ 10 8 . The amount of TAGs in 10 8 cells was defined as the quantitative lipid-accumulation ability.
6
Quantitative HPLC Analysis of Oxalic Acid
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The extracts in the volumetric flasks were filtered through a cellulose acetate syringe filter with a pore size of 0.45 μm (dismic‐25cs, Advantec, CA, USA) into 1 ml glass vials. The samples were analyzed with a high‐performance liquid chromatography (HPLC) system, using a 300 mm × 7.8 mm Rezex ion exclusion column (Phenomenex Inc., Torrance, CA, USA) attached to a Cation‐H guard column (Bio‐Rad, Richmond, CA, USA) held at 25°C. Analysis was performed by injecting 20 μl of sample or standard onto the column using an aqueous solution of 25 mM sulfuric acid (HPLC grade Baker Chemicals, Phillipsburg, NJ, USA) as the mobile phase, pumped isocratically at 0.6 ml/min, with peaks detected at 210 nm. The HPLC equipment consisted of a Shimadzu LC‐10AD pump, CTO‐10A column oven, SPD‐10Avp UV–vis detector (Shimadzu, Kyoto, Japan) and a Waters 717 plus auto‐sampler (Waters, Milford, MA, USA). Data acquisition and processing were undertaken using a PeakSimple Chromatography Data System (model 203) and PeakSimple software version 4.37 (SRI Instruments, Torrance, CA, USA). The oxalic acid peak was identified by comparing the retention time to a standard solution, and by spiking an already‐filtered sample containing a known amount of oxalic acid standard. The insoluble oxalate content of each sample was calculated by difference between the total and the soluble oxalate contents.
7
Propagation and Characterization of Phages
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Bacteriophage MS2 (NBRC 102619, National Institute of Technology and Evaluation (NITE), Tokyo, Japan) was propagated as described elsewhere (Torii et al., 2019 (link)). Pseudomonas phage φ6 (NBRC 105899, NITE) was propagated using Pseudomonas syringae (NBRC14084, NITE) as a host strain. Briefly, 3 mL of phosphate buffer (PB) (10 mM, pH 7.0) was added on the soft LB plates (0.7% agar) semiconfluent with φ6 plaques (approximately 1000 plaques/plates) and incubated at room temperature for 5 h. The suspension and soft agar were then removed from the plate and transferred to a centrifuge tube. Next, 10 mL of recovered viruses were clarified by centrifugation at 3500g for 15 min and filtered through a cellulose acetate filter (0.2 μm, DISMIC-25CS, Advantec, Tokyo, Japan). MNV S7-PP3 strain was propagated using RAW.264.7 cell as a host (Kitajima et al., 2008 (link)). The concentrations of propagated stocks were approximately 1011 PFU/mL, 5 × 109 PFU/mL, and 1011 copies/mL for MS2, φ6, and MNV, respectively. The propagated stocks were stored at 4 °C. Before the primary concentration, the MS2 and φ6 stocks were diluted by 1000-fold and 100-fold, respectively, to prepare the diluted stocks.
8
Isolation of Phages from Pig Fecal Samples
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Fecal samples were obtained from a pig farm in Hongseong-gun, Chungcheongnam-do,
Korea, suspended in 50 mL SM buffer (50 mM Tris-HCl [pH 7.5], 0.1 M NaCl, and 8
mM MgSO4·7H2O), and centrifuged at 4,000×g
for 10 min. The supernatant was filtered using a 0.45 μm membrane filter
(Dismic®-25CS, Advantec, Japan) to prepare a sample
solution. Next, 18 mL supernatant and 2 mL ETEC FC02 bacterial cell culture
(OD540=0.4) were mixed and incubated at 37°C for 18
h. After centrifugation at 4,000×g for 10 min, the supernatant was
filtered using a 0.45 μm membrane filter. Then, 100 μL filtered
supernatant was mixed with the host bacteria E. coli FC02
bacterial cell culture and 5 mL Luria-Bertani (LB) broth containing 0.7%
soft agar. The mixture was poured into 1.5% LB agar and incubated
overnight at 37°C. Individual single plaques were harvested from the agar
plate using sterile pipette tips, resuspended in 500 μL SM buffer, and
the double-layer agar technique was repeated (Šimoliūnas et al., 2014 (link)).
Korea, suspended in 50 mL SM buffer (50 mM Tris-HCl [pH 7.5], 0.1 M NaCl, and 8
mM MgSO4·7H2O), and centrifuged at 4,000×g
for 10 min. The supernatant was filtered using a 0.45 μm membrane filter
(Dismic®-25CS, Advantec, Japan) to prepare a sample
solution. Next, 18 mL supernatant and 2 mL ETEC FC02 bacterial cell culture
(OD540=0.4) were mixed and incubated at 37°C for 18
h. After centrifugation at 4,000×g for 10 min, the supernatant was
filtered using a 0.45 μm membrane filter. Then, 100 μL filtered
supernatant was mixed with the host bacteria E. coli FC02
bacterial cell culture and 5 mL Luria-Bertani (LB) broth containing 0.7%
soft agar. The mixture was poured into 1.5% LB agar and incubated
overnight at 37°C. Individual single plaques were harvested from the agar
plate using sterile pipette tips, resuspended in 500 μL SM buffer, and
the double-layer agar technique was repeated (Šimoliūnas et al., 2014 (link)).
9
Purification of Female Bioactive Compounds
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Eight sexually developed females were kept in a container filled with 800 mL tap water for 48 hours. The water was then filtered using Whatman filter paper and the filtrate was lyophilised. The lyophilised sample was dissolved in 50 mM Tris-HCl (8.5 mL) containing 150 mM NaCl (pH 7.5) and filtered again using 0.45-μm-pore filters (DISMIC-25CS, Advantec). One quarter of the total amount of water was saved while the remainder was used for affinity chromatography.
10
Synthesis of Fibrin-Targeted GC-AuNPs
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GC (300 mg) was dissolved in 300 mL of water (0.1 wt/wt %) and stirred for over 24 hours. The GC solution was filtered with 0.2 μm disposable syringe filter (DISMIC-25cs; Advantec, Tokyo, Japan) and boiled to 70oC before 100 mL of HAuCl4·3H2O solution (1 mM) was added quickly. The solution was kept at 70oC under stirring until the color of solution changed from light yellow to deep red in 8 hours. After another 16 hours, the heat source was removed and the GC-AuNP colloidal solution was cooled down to room temperature. Then, fibrin-targeting peptides used in the EP-2104R11 (link) (3.5 mg) were dissolved in 0.4 mL dimethyl sulfoxide (Sigma-Aldrich) with 1-ethyl-3-(3-dimethylaminopropyl) carbodi carbodiimide hydrochloride (Sigma-Aldrich, 0.5 mg) and n-hydroxy succinimide (0.5 mg; GE Healthcare, Piscataway, NJ), while being vigorously stirred for 30 minutes. After 15 minutes, the peptide solution was mixed with 15 mL of GC-AuNP colloid (3 mg Au per mL), and stirred for 4 hours. Then, fib-GC-AuNPs were centrifuged (9000 rpm, 45 minutes) and washed three times with water to remove remnants of the peptide.
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