All physiological experiments were performed in triplicate. The effects of temperature, pH, and concentrations of NaCl on cell growth, utilization of carbohydrates and sensitivity to antibiotics were determined by hydrogen production in gas phase of cultures. Hydrogen and methane in gas phase of cultures were measured with a gas chromatography equipped with a thermal conductivity detector (GC-8A; Shimadzu, Japan). Glucose, acetate, and ethanol in liquid phase of cultures were measured with a high-performance liquid chromatography (HPLC) (LC20; Shimadzu) with Shim-pack SPR-H column (Shimadzu) or HPLC (LC-2000Plus, Jasco) with Aminex HPX-87H column (BIO-RAD). Methyl esters of cellular fatty acids were identified and quantified via a gas chromatography-mass spectrometry (M7200A GC/3DQMS system; Hitachi).
Lc 2000plus
Manufactured by Jasco
Sourced in Japan
The LC-2000Plus is a high-performance liquid chromatography (HPLC) system designed for analytical and preparative applications. It features a compact, modular design and includes a quaternary solvent delivery system, an autosampler, and a UV/Vis detector. The LC-2000Plus is capable of delivering accurate and precise flow rates for a wide range of HPLC applications.
Automatically generated - may contain errors
Lab products found in correlation
Rezex rhm, by Phenomenex (3 mentions)
Uv 2075, by Jasco (2 mentions)
5c18 ms 2, by Nacalai Tesque (2 mentions)
Lc 20, by Shimadzu (1 mentions)
Gc 8a, by Shimadzu (1 mentions)
Hesperidin, by Tokyo Chemical Industry (1 mentions)
Methanol, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Polystyrene standards, by Polymer Laboratories (1 mentions)
Plgel columns, by Agilent Technologies (1 mentions)
Ri 2031 plus, by Jasco (1 mentions)
As 2055plus autosampler, by Jasco (1 mentions)
Fp 2025, by Jasco (1 mentions)
Uv 2070 detector, by Jasco (1 mentions)
Bovine gamma globulin, by Bio-Rad (1 mentions)
Bovine thyroglobulin, by Bio-Rad (1 mentions)
Myoglobin, by Bio-Rad (1 mentions)
Vitamin b12, by Bio-Rad (1 mentions)
Pu 2880 plus injector, by Jasco (1 mentions)
Pda md 2018 detector, by Jasco (1 mentions)
22 protocols using lc 2000plus
1
Physiological Impacts on Microbial Metabolism
2
HPLC Analysis of Fermentation Byproducts
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Glucose, gluconic acid, cellobiose, xylose, arabinose, hydroxymethyl furfural and the furfural content of the culture media were determined by HPLC using a Rezex RHM (Phenomenex, Torrance, CA, USA) column with isocratic elution (flow rate of 0.400 mL/min and mobile phase of 0.025 M H2SO4), a column oven set at 45 °C and a refractive index detector (RI) (LC 2000 plus, Jasco, Tokio, Japan). The RI detector was used for the analysis of glucose, cellobiose, xylose and arabinose. Gluconic acid, furfural and HMF were detected spectrophotometrically with DAD. Gluconic acid was detected at 220 nm, and furfural and HMF were detected at 275 nm [21 (link),22 (link),23 (link)]. The analysis was performed in triplicate by taking 3 Petri dishes each day until the end of the incubation (10 days).
3
Hesperidin Extraction and Quantification
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Hesperidin was purchased from Tokyo Chemical Industry (Tokyo, Japan). Trifluoroacetic acid (TCA) and methanol were purchased from Sigma (St. Louis, MO, USA). For the preparation of GME, unripe fruit of Citrus unshiu was obtained from the orchards of Jeju Island (Korea) and extracted with ethanol two times. Briefly, the dried unripe fruit was extracted with 4 L of 70% aqueous ethanol three times at 80 °C for 1 h, followed by further extraction with 95% aqueous ethanol to yield 90% ethanol-insoluble precipitate (GME). For analysis of the Hesperidin content in GME, an HPLC (LC-2000Plus, Jasco International, Tokyo, Japan) assay using a Luna® C18 5 μm column (4.6 × 250 mm) was performed. A gradient elution was achieved with various ratios of solvent A (0.1% TCA in water) to solvent B (methanol), with a flow rate of 1 mL/min as follows: 0–25 min, 70:30, 26–30 min, 10:90, and 31 min onward, 70:30. The wavelength of acquisition was set to 280 nm. The average content of Hesperidin in the GME was 35% (Supplemental Fig. 1).
4
Polymer Molecular Weight Analysis
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Weight-average (Mw) and number-average molecular weight (Mn) values and molecular weight distributions (Mw/Mn) values of the polymers were evaluated using a Jasco LC-2000Plus gel permeation chromatograph (GPC) equipped with a refractive index detector RI-2031Plus (Jasco, Oklahoma City, OK, USA) using 3 Agilent (Santa Clara, CA, USA) PLgel columns, 5 × 10−6 m particle size, 300 × 7.5 mm (Mw range: 5 × 102 to 17 × 105 g·mol−1). THF was chosen as eluent at a flow rate of 0.5 mL·min−1 at 35 °C. The GPC samples were injected using a Jasco AS-2055Plus autosampler. The instrument was calibrated using polystyrene standards from 580 to 3,250,000 Da (Polymer Laboratories, Church Stretton, UK). Probes have been run in duplicates, showing full consistency and overlapping of the data.
5
Serum Neopterin Assessment for Macrophage Activity
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The serum level of neopterin, an activation marker produced primarily by IFN-γ-stimulated monocytes and macrophages (68 (link),69 (link)), was assessed for macrophage activity. Determination of serum neopterin was outsourced to SRL, Inc. Serum (0.3 ml) was analyzed by a reverse-phase high-performance liquid chromatography column-switching method (LC-2000Plus; JASCO Corporation) (70 (link)) using Wakosil GP-N6 4.6x150 mm as a pretreatment column and Wakosil-II 5C18 HG 4.6x250 mm as an analysis column (FUJIFILM Wako Pure Chemical Corporation). The neopterin level was determined by detecting its native fluorescence (excitation, 353 nm; emission, 438 nm) with a fluorescence detector (FP-2025; JASCO Corporation).
6
Analysis of Silage Fermentation Products
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The products of the silage fermentation were analysed using the analysis method for fermented feed described by Cai [22 ]. The fermented feed sample (10 g) was homogenized in 90 mL of sterilized distilled water and placed in a refrigerator at 4 °C for 24 h. After evenly mixing, the extract was filtered through a quantitative ashless filter paper (5A, 110 mm; Advantec Co., Ltd., Tokyo, Japan). The pH value was determined by a glass electrode pH meter (D-71; Horiba Co., Ltd., Kyoto, Japan).
The ammonia nitrogen (NH3-N) content of the fermented feed was analysed using the Kjeltec auto distillation system (2200; Foss Tecator, Höganäs, Sweden), as described by Cai [22 ]. The fermented feed filtrate was shaken through a cation exchange resin (Amberlite, IR 120B H AG; Organo Corporation, Tokyo, Japan) and centrifuged at 6500×g for 5 min at 4 °C. The supernatants were passed through a 0.45 µm filter (DISMIC 13HP; Toyo Roshi Kaisha, Ltd, Tokyo, Japan), and the filtrates were injected into an HPLC system (LC-2000 plus; JASCO Corporation, Tokyo, Japan) to determine the organic acid content in accordance with the feed analysis method described by Cai [22 ].
The ammonia nitrogen (NH3-N) content of the fermented feed was analysed using the Kjeltec auto distillation system (2200; Foss Tecator, Höganäs, Sweden), as described by Cai [22 ]. The fermented feed filtrate was shaken through a cation exchange resin (Amberlite, IR 120B H AG; Organo Corporation, Tokyo, Japan) and centrifuged at 6500×g for 5 min at 4 °C. The supernatants were passed through a 0.45 µm filter (DISMIC 13HP; Toyo Roshi Kaisha, Ltd, Tokyo, Japan), and the filtrates were injected into an HPLC system (LC-2000 plus; JASCO Corporation, Tokyo, Japan) to determine the organic acid content in accordance with the feed analysis method described by Cai [22 ].
7
Silage Fermentation Product Analysis
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The fermentation products of the silages were analyzed using the cold water extract method (30 ). The 10 g of silage sample was homogenized in 90 mL of sterilized distilled water and kept in a 4°C refrigerator for 24 h. Thereafter, the extract samples were filtered through quantitative ashless filter paper (circle size: 5A, 110 mm, Advantec Co., Ltd., Tokyo, Japan). The filtrate was used to determine pH, NH3-N, and organic acid including lactic acid, acetic acid, propionic acid, and butyric acid. The pH was measured using a pH meter (D-71, Horiba Co., Ltd., Kyoto, Japan). The steam distillation of the filtrates was used to determine NH3-N using the Kjeltech auto distillation (2200, Foss Tecator, Hoganas, Sweden) as described by Cai (28 ). The filtrates of silages were injected into an HPLC system (LC-2000 plus, JASCO Co., Tokyo, Japan) to determine organic acid (30 ). The HPLC system was equipped with a Shodex RSpak column (KC-811, Showa Denko K. K., Tokyo, Japan), 60°C oven, Jasco UV-2070 detector (450 nm), 3 mM HClO4 eluent and reagent (0.2 mM bromothymol blue + 8 mM Na2HPO4 + 2 mM NaOH), and 1.0 mL/min flow rate.
8
Quantification of Zearalenone Residues
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All isolates were inoculated at 1% (v/v) in TSB containing 5 mg L−1 ZEN for 24 h at 37 °C. Then, samples were centrifuged for 20 min at 8000× g at 4 °C; the supernatants were collected and extracted with an equal volume of chloroform and sonicated for 30 min. The organic phase was separated by centrifugation (500× g for 10 min at 25 °C) and dried with nitrogen gas at 63.5 °C. The residues were re-dissolved in 1 mL methanol and concentrated to 1/5 of the original volume by centrifugal vacuum concentrator (5301 VacuFuge, Eppendorf®, Hamburg, Germany) at 60 °C, then filtered through a 0.22 μm nylon syringe filter before loaded into HPLC (LC-2000Plus, JASCO, Tokyo, Japan) with a fluorescence detector (excitation and emission wavelengths were 274 and 440 nm) and the Luna® 5 μm C18(2) 100-Å, LC column (250 × 4.6 mm) (Phenomenex, Torrance, CA, USA) to detect residual ZEN. The mobile phase was acetonitrile solution (50:50, v/v).
9
Quantification of β-carotene in C2C12 myotubes
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C2C12 myotubes in 35-mm dishes were cultured in differentiation medium in the presence of 10 μM β-carotene for 12 h at 37°C or 4°C as a negative metabolic control. Cells were then washed three times with PBS. Cells were sonicated in 0.1 ml of sucrose buffer (20 mM Hepes-NaOH, pH 7.5, containing 250 mM sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM DTT) containing 100 pmol of β-cryptoxanthin (an internal standard) and centrifuged at 15,000 × g for 15 min. The supernatant was mixed with 0.2 ml of a sucrose buffer. Samples were extracted three times with 0.3 ml of hexane, dried under nitrogen, and reconstituted in the appropriate mobile phase [methanol:acetonitrile:chloroform = 47:47:6 (v/v/v)]. The samples were analyzed by HPLC with LC-2000Plus (JASCO Corporation, Tokyo, Japan) equipped with a UV/visible dual beam detector (UV-2070 Plus). The column was a 4.6 × 250 mm Ultrasphere COSMOSIL Packed Column 5C18-AR-II (Nacalai Tesque). Carotenoids were eluted by an isocratic mobile phase [methanol:acetonitrile:chloroform = 47:47:6 (v/v/v)] at a flow rate of 1 ml/min at 40°C and monitored by measuring absorbance at 450 nm.
10
Protein Size Analysis by SEC-HPLC
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Size-exclusion chromatography was performed by analytical HPLC (Jasco LC-2000 Plus) equipped with a PU 2880 Plus injector and a PDA MD 2018 detector (Jasco). The samples (50 μM in 100 mM Tris-HCl at pH 7.4) were separated by a system containing a Phenomenex BioSep-SEC-S3000 column (7.8 × 300 mm, 5 μm, resolution range of 15 to 2000 kDa, Phenomenex, Inc., Torrance, California, USA) using a flow rate of 1.0 mL/min in 100 mM Tris-HCl buffer (pH 7.4) and 50 mM NaCl. The elution profile was monitored by absorbance (λ = 280 nm). Bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa) were used as molecular weight standards (Bio-Rad). The chromatograms were analyzed using Jasco BORWIN, version 1.50, software (Jasco). The enzymes were reduced or oxidized by treatment with 5 mM TCEP or 1.2 molar excess of H2O2, respectively, for 30 min at 25 °C.
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