Measurements of dopamine, noradrenalin and serotonin levels were performed by HPLC analysis, as previously described [31 (link)]. Briefly, colonic tissue was homogenized and then centrifuged at 4°C (14000g; 10min). The remaining solution was filtered and injected (20 µl/injection) into the HPLC system (Shimadzu LC prominence). Separation of each neurotransmitter was performed with the aid of a reverse phase analytical column (Waters Symmetry 300C18). The mobile phase consisted of a 10% MeOH solution (pH 4) containing 70 mM KH2PO4, 1 mM octanesulfonic acid and 1 mM and was delivered at a rate of 1 ml/min. A coulometric electrochemical detector (ESA Coulochem III) was used for signal detection. The first and the second electrode of the analytical cell were set at +50 mV and +350 mV, respectively, and the guard cell was set at -100 mV. Data were processed with the Shimadzu LC solution software and were expressed as picogram per milligram of wet tissue.
Prominence lc
Manufactured by Shimadzu
Sourced in Japan, United States
The Prominence LC is a high-performance liquid chromatography (HPLC) system manufactured by Shimadzu. It is designed to provide reliable and efficient separation and analysis of a wide range of compounds. The Prominence LC features advanced technology and components to ensure accurate and reproducible results.
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Lab products found in correlation
Tolbutamide, by Merck Group (4 mentions)
Carbopac pa1, by Thermo Fisher Scientific (4 mentions)
N benzylbenzamide, by Merck Group (4 mentions)
Xdb c18 column, by Agilent Technologies (4 mentions)
Waters symmetry 300c18, by Waters Corporation (3 mentions)
Lc solution software, by Shimadzu (3 mentions)
Rezex rhm monosaccharide h 8, by Phenomenex (3 mentions)
Ics 3000 hplc system, by Thermo Fisher Scientific (2 mentions)
4000 qtrap, by Shimadzu (2 mentions)
3200 qtrap, by Shimadzu (2 mentions)
4000qtrap mass spectrometer, by Shimadzu (2 mentions)
Agilent xdb c18 column, by Agilent Technologies (2 mentions)
Symmetry300 c18, by Waters Corporation (1 mentions)
Rid 10a, by Shimadzu (1 mentions)
Esa coulochem 3, by Thermo Fisher Scientific (1 mentions)
Malachite green phosphate assay kit, by Merck Group (1 mentions)
Spd 20a, by Shimadzu (1 mentions)
Tskgel deae 5pw, by Tosoh (1 mentions)
Avance 3 700, by Bruker (1 mentions)
Avance 3 400 mhz, by Bruker (1 mentions)
28 protocols using prominence lc
1
Measuring Neurotransmitter Levels in Colon Tissue
2
Quantification of Striatal Dopamine Metabolites
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Dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) were measured in the striata by high-performance liquid chromatography (HPLC) analysis, as previously described [28 ]. Briefly, rat striatal tissue was homogenized and centrifuged (14,000×g for 20 min at 4 °C). Then, the supernatant fractions were filtered and injected directly into the HPLC (Shimadzu LC prominence, Shimadzu Corporation, Kyoto, Japan). DA, DOPAC, and HVA were separated on a reverse phase analytical column (Waters Symmetry300C18; Waters, Milford, MA). The mobile phase (pH 4) consisted of 10% MeOH, 70 mM KH2PO4, 1 mM octanesulfonic acid, and 1 mM EDTA and was delivered at a rate of 1 ml/min. Detection was performed with a coulometric electrochemical detector (ESA Coulochem III; ESA, Chelmsford, MA). Data were acquired and processed with the Shimadzu LC solution software (Shimadzu Corporation) and were expressed as nanogram per milligram of wet tissue. The DOPAC/DA ratio was calculated for each animal as an index of the DA turnover.
3
Monoamine Quantification in Mouse Brain
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In WT and KOR null mice, the hypothalamus, prefrontal cortex and striatum were dissected on an ice-cold plate, and immediately frozen on dry ice and stored at −80 °C until analysis. Then tissue was homogenized and then centrifuged (14,000×g; 10 min at 4 °C). The remaining supernatant fraction was filtered and injected (20 μl/injection) into the High-Performance Liquid Chromatography (HPLC) system (Shimadzu LC Prominence; Shimadzu Corporation; Kyoto, Japan)56 (link),57 (link). Dopamine and its metabolite, DOPAC, 5-HT and NE were separated using a reverse phase analytical column (Waters Symmetry 300C18; Waters, Milford, MA, US). The mobile phase consisted of a 10% MeOH solution (pH = 4) containing 70 mM KH2PO4, 1 mM octanesulfonic acid and 1 mM EDTA, and was delivered at a rate of 1 ml min−1. Detection was performed with a coulometric electrochemical detector (ESA Coulochem III; ESA, Chelmsford, MA). Data were processed with the Shimadzu LC Solution Software (Shimadzu Corporation; Kyoto, Japan) and were expressed as ng mg−1 of wet tissue.
4
Molecular Weight Distribution Analysis of OVDs
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The molecular weight distribution of sample was determined by high-performance liquid chromatography (LC Prominence; Shimadzu Co., Ltd., Kyoto, Japan) using a size exclusion column (OHpak SB-806M HQ; Shodex, Co., Ltd., Tokyo, Japan) and a reflective index detector (RID-10A; Shimadzu Co., Ltd.). The flow rate was 0.3 mL min−1 of 0.5 M NaCl at 35 °C. Each sample was diluted 300 times by the eluent and aliquots (100 μL) before injection. The initial molecular weight distribution and viscosity of each OVD were independent of additives. The elution profiles of the basal OVD and OVD containing D-sorbitol are shown in Fig. 5 . Relative weight-average molecular weight (Mw) was calculated by using 6 Pullulan standards for molecular weight (Shodex, Co., Ltd.).![]()
Initial molecular weight distribution of OVDs with different D-sorbitol contents. SEC-HPLC elution profiles obtained with (—) basal OVD, (---) OVD with 0.5% D-sorbitol, and (•••) OVD with 1.0% D-sorbitol. The peaks detected at 20–26 min and 26–31 min correspond to HA and CS, respectively.
5
Neurotransmitter Analysis in BAT Tissue
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BAT samples were dissected on a chilled plate, and immediately flash-frozen in dry ice before being stored at −80 °C until further analysis. The frozen tissue was subsequently homogenized, sonicated, and centrifugated at 14,000 g for 20 min at 4 °C. The resulting supernatant fraction was filtered and then injected into a high-performance liquid chromatography (HPLC) system (Shimadzu LC Prominence; Shimadzu Corporation) [27 ,28 (link)]. To separate norepinephrine (NE), dopamine (DA), and serotonin (5-HT), a reverse-phase analytical column (Waters Symmetry 300C18; Waters) was employed. The mobile phase consisted of a 10% MeOH solution (pH = 4) containing 70 mM KH2PO4, 1 mM octanesulfonic acid, and 1 mM EDTA, delivered at a flow rate of 1 ml/min. Detection of the neurotransmitters was achieved using a coulometric electrochemical detector (ESA Coulochem III; Thermo Scientific). The first and second electrodes of the analytical cell were set at +50 mV and +350 mV, respectively, while the guard cell was set at −100 mV. Data acquisition and processing were performed using the Shimadzu LC solution software (Shimadzu Corporation). The concentrations of the neurotransmitters were expressed as pg/mg of wet tissue, as shown [27 ,28 (link)].
6
Analytical Methods for Biomass Components
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The total amounts of glucan and xylan were analyzed as described previously (Shiroma et al., 2011 (link)). Phosphorus was defined in this study as phosphorus atoms in solubilized phosphate and measured using a Malachite Green Phosphate Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) with phosphoric acid as a standard. The calcium ions in the solutions liberated in acidic conditions with HCl were measured using a Metalloassay Calcium LS (Metallogenics Co., Ltd., Chiba, Japan) with CaCl2 as a standard. Potassium ions in the solutions were measured using a pH/ion meter (F-53, HORIBA, Ltd. Kyoto, Japan) equipped with a potassium electrode (#6582, HORIBA, Ltd.) with KCl as a standard. p-Coumarate, ferulate, acetic acid and lactic acid were measured with the corresponding standards using an isocratic HPLC system (LC Prominence, Shimadzu Corporation, Kyoto, Japan). An ODS column (CAPCELL PACK C18 MGII, Osaka Soda Co., Ltd., Osaka, Japan) and a UV detector (SPD-20A, Shimadzu Corporation, Kyoto, Japan) were used for the measurements of p-coumarate and ferulate (mobile phase:20% acetonitrile in 0.1% H3PO4, λ = 310 nm). Acetic acid and lactic acid were measured by using an Aminex HPX-87H column (Bio-Rad Laboratories Co., Ltd., Tokyo, Japan, mobile phase: 5 mM H2SO4) with a refractive index detector (RID-16A, Shimadzu Corporation, Kyoto, Japan).
7
Monitoring bacterial growth and substrate utilization
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Cell growth was monitored by measuring the optical density at 600 nm wavelength (OD 600 ) in a spectrophotometer (Uvikon 860, Z€ urich, Switzerland). At different time intervals, samples were removed for growth measurement and substrate utilization analysis. Samples were centrifuged (8000 3 g for 10 min) and the supernatant was applied to HPLC analysis for quantification of benzoate, o-phthalate, nitrate and nitrite (data not shown). Fifty microliter of sample supernatant was injected into an HPLC (LC prominence Shimadzu, Japan) equipped with a Grom TM Sil 120 ODS-5 ST, column (4 mm 3 150 mm, 5 mm) using an UV detector (SPD-M20A prominence) at 230 nm for signal detection. Eluent A (acetonitrile) and eluent B (20 mM H 3 PO 4 ) were used at a flow rate of 0.8 ml/min at 308C. A gradient of eluent B was applied for 30 min: 5 min isocratic of 92%; 3 min gradient to 89%; 10 min gradient to 80%; 5 min isocratic of 80%; 2 min gradient to 95%; 3 min gradient to 92%; 2 min isocratic 92%.
8
Purification of Native Biglycan Proteoglycan
9
NMR and Mass Spectrometry Analysis
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NMR spectra were recorded on a Bruker Avance III 400 MHz (399.87 MHz for 1H, 100.55 MHz for 13C, D2O, 30 °C, 6 ) and a Bruker Avance III 700 MHz spectrometer (700.13 MHz for 1H, 176.05 MHz for 13C, D2O and MeOD (9:1 [v/v]), 30 °C, 7 /8 ). The residual signal of water was used as an internal standard (δH 4.732 ppm); carbon chemical shifts were referenced to acetone (δC 30.50 ppm). The following NMR experiments were performed using the manufacturer’s software: COSY, 1H-13C HSQC, HSQC-TOCSY, 1D-TOCSY and 1H-13C HMBC. Mass spectra were recorded with a Shimadzu Prominence LC analytical system (Shimadzu, Japan) as described previously (Kotik et al. 2021 (link)).
10
Quantitative Analysis of Biomass Hydrolysis
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The concentration of monomeric sugars was determined with a high-pressure liquid chromatography (HPLC) system (Dionex ICS-3000). The concentration of monomeric sugars, ethanol and degradation products, such as acetic acid, furfural and 5-hydroxymethylfurfural (5-HMF) were measured using refractive index detection on a Shimadzu Prominence LC, as described by Suko and Bura [19 (link)]. Monomeric and oligomeric soluble carbohydrates were determined using NREL LAP TP-510-42623 [18 ]. Phenolic concentration in the liquid fraction was assayed by the Folin–Ciocalteu method [20 ], using a ultra-violet (UV) spectrophotometer (Shimadzu, Tokyo, Japan) at 765 nm. Gallic acid was used as calibration standard.
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