In gramicidin perforated experiments and in experiments performed for assessing the KCC2 efficacy, GABAAR responses were isolated by using a cocktail of drugs containing 0.2 μM tetrodotoxin (TTX, Latoxan Laboratory, France), 4 mM kynurenic acid (Millipore Sigma), 10 μM (+)-tubocurarine (Millipore Sigma), 5 μM Dihydro-β-erythroidine hydrobromide (DHβE, Bio-techne, France), and 3 μm strychnine (Millipore Sigma) that respectively blocked voltage-dependent Na+ action potentials, and glutamate, cholinergic, and glycinergic input to MNs. In CsCl experiments, IPSCs were isolated pharmacologically using DL-AP5 40 μM ((2R)-amino-5-phosphonovaleric acid, Bio-techne, France) and CNQX 20 μM (6-cyano-7-nitroquinoxaline-2,3-dione, Bio-techne, France). mIPSCs were isolated in the presence of 0.2 μM TTX (Latoxan, France). GABA and glycine mIPSCs were isolated by adding 3 μM strychnine or 3 μM GABAzine (SR 95531 hydrobromide, Bio-techne, France), respectively. These blockers were added to the cocktail containing 0.2 µM TTX, 4 mM kynurenic acid, 10 μM (+)-tubocurarine and 5 μM DHβE. In experiments assessing the KCC2 efficacy, 10 µM bumetanide (Millipore Sigma) was applied to block NKCC1 and 10 µM VU0240551 (Bio-techne, France) (10 µM) to block KCC2. Serotonin (5-HT, 10 µM), dopamine (DA, 100 µM) and N-Methyl-D-aspartic acid (NMDA, 10 µM) were from Millipore Sigma.
Kynurenic acid
Manufactured by Merck Group
Sourced in United States, Germany, France, Italy, United Kingdom
Kynurenic acid is a chemical compound that is commonly used in laboratory research. It is a metabolite of the amino acid tryptophan and is known to have various biological functions. The core function of kynurenic acid is to serve as a biochemical tool for scientific investigation and analysis, particularly in the fields of neuroscience and immunology.
Automatically generated - may contain errors
Lab products found in correlation
Picrotoxin, by Merck Group (16 mentions)
Kynurenine, by Merck Group (11 mentions)
Xanthurenic acid, by Merck Group (9 mentions)
Quinolinic acid, by Merck Group (7 mentions)
Hyaluronidase, by Merck Group (6 mentions)
Mk 801, by Merck Group (6 mentions)
L tryptophan, by Merck Group (6 mentions)
Strychnine, by Merck Group (5 mentions)
Tryptophan, by Merck Group (5 mentions)
Sr95531, by Bio-Techne (5 mentions)
Picrotoxin, by Bio-Techne (5 mentions)
3 hydroxyanthranilic acid, by Merck Group (5 mentions)
Anthranilic acid, by Merck Group (5 mentions)
L kynurenine, by Merck Group (5 mentions)
Formic acid, by Merck Group (5 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (5 mentions)
Trypsin, by Merck Group (4 mentions)
D glucose, by Merck Group (4 mentions)
3 hydroxykynurenine, by Merck Group (4 mentions)
Poly d lysine, by Merck Group (4 mentions)
134 protocols using kynurenic acid
1
Isolating GABAAR Responses for Functional Analysis
2
Tryptophan Metabolism Metabolite Analysis
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Methanol (CH3OH), ammonium acetate (CH3COONH4), acetonitrile (CH3CN), picolinic acid, 3-hydroxykynurenine, quinolinic acid, serotonin, 5-hydroxytryptophan, kynurenine, 3-hydroxyanthranilic acid, tryptamine, L-tryptophan, 5-hydroxyindole, acetic acid, indoxyl sulfate, N-Acetylserotonin, xanthurenic acid, indole-3-acetamide, kynurenic acid, DL-indole-3-lactic acid, indole-3-carboxaldehyde, indole-3-acetic acid, tryptophol, melatonin, 5-hydroxyindole acetic acid-D5, serotonin-D4, indole-3-lactic acid-D4, kynurenic acid-D5, melatonin-D4, picolinic acid-D3, tryptamine-D4, xanthurenic acid-D4, 3-hydroxyanthranilic acid-D3, 3-hydroxykynurenine-13C2-15N, 5-hydroxytryptophan-D4, indole-3-acetamide-D5, kynurenine-D4, L-tryptophan-13C11,15N, and tryptophan-D5 were purchased from Sigma Aldrich, Merck KGaA (Darmstadt, Germany) and other reagents and chemicals used in the study were purchased from local suppliers. The reagents and chemicals were of analytical grade or higher purity. Additionally, the yeast strain S. cerevisiae STG S101 was purchased from Fermentis (Marcq-En-Baroeul, France).
3
Isolation of Neural Progenitor Cells
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NPCs were isolated from periventricular dissection of the adult YFP+ mouse as previously described (Morshead et al., 2002 (link); Babona-Pilipos et al., 2011 (link), 2012 ). Briefly, the isolated tissue was enzymatically dissociated in hyaluronidase (1157 units/ml, Millipore-Sigma), trypsin (1.33 mg/ml, Millipore-Sigma) and kynurenic acid (0.13 mg/ml, Millipore-Sigma) for 25 min and mechanically dissociated through trituration. The solution was spun down and resuspended in trypsin inhibitor (0.33 mg/ml, Worthington Biochemical Corporation). The suspension was washed with serum-free media (SFM; 1× DMEM/F12, 0.6% glucose, 0.1% NaHCO3, 5 mM HEPES buffer, glutamine, defined hormone and salt mixture, and penicillin/streptavidin). The cells were plated in SFM with epidermal growth factor (20 ng/ml; Millipore-Sigma), basic fibroblast growth factor (10 ng/ml; Millipore-Sigma), and heparin (2 μg/ml, Millipore-Sigma) in T25 flasks. The neural precursor colonies (neurospheres) were collected, mechanically dissociated and replated in growth media (passaged) every 6–7 d.
4
Fecal and Urine Metabolomic Analysis
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The fecal water obtained for LPS quantification was also used for metabolomics experiments. Urine samples were prepared according to the procedure described by Marques et al.43 (link). Samples were analyzed by HPLC/Orbitrap according to the method described by Fernandes et al.44 (link). Mass spectrometry (MS) data was uploaded into XCMS Online and was processed as a multi-group experiment using the default HPLC/Orbitrap parameters in negative mode (fecal samples) or positive mode (urine samples)45 (link). Isotopes and adducts were annotated using CAMERA and arranged into feature groups. Metabolite features were selected to assess the differences between the fecal and urine samples of C, C + BE, HF and HF + BE groups. Tryptophan, kynurenine and kynurenic acid (MilliporeSigma, St. Louis, MO, USA) were used as standards.
5
Pharmacological Isolation of GABAAR Responses
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In the gramicidin perforation experiments, GABAAR responses were isolated by using a cocktail of drugs containing 0.2 µM tetrodotoxin (TTX, Latoxan Laboratory, France), 4 mM kynurenic acid (Millipore Sigma), 10 µM (+)-tubocurarine (Millipore Sigma), 5 µM dihydro-β-erythroidine hydrobromide (DHΒE, Bio-Techne, Lille, France), and 3 µM strychnine (Millipore Sigma), which blocked voltage-dependent Na+ action potentials and glutamate, cholinergic, and glycinergic inputs to MNs, respectively. VU0240551 (Bio-Techne, France) (10 µM) was applied to specifically block KCC2. Serotonin (5-HT, 10 µM) was purchased from Millipore Sigma. Methysergide maleate (10 µM) and ketanserin tartrate (10 µM) (Bio-techne) were used to evaluate the involvement of 5-HTRs in the effect of 5-HT. Combined, these antagonists are known to efficiently block 5-HT2A-CR [31 (link),32 (link)].
6
Colorimetric Assay for Endogenous XA
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Colorimetric assay was used to determine the endogenous XA.40 (link) Briefly, 8 mM solution of N, N-diethyl-p-phenylenediamine (DE-PPD) (ThermoScientific, USA), 300 IU/mL horseradish peroxidase (POD) (ThermoScientific, USA), XA or kynurenic acid (KA) (Sigma, USA) standards (2.5–150 μM), 0.1M acetate buffer (pH 4.3) were prepared with water. Unfed uninfected or A. phagocytophilum-infected nymphal ticks or tick cells were homogenized in water and supernatants were used as test samples. Infection was confirmed with qPCR using primers specific for A. phagocytophilum. For tick cell line experiments, 2 × 105 tick cells were seeded in L15B300 medium on to 12 well plates and incubated for 24 h. After incubation, one group of tick cells were infected with A. phagocytophilum, and other group was retained as uninfected controls. Tick cell samples were prepared after 48 h p.i. To 187.5 μL of 0.1M acetate buffer (pH 4.3), 46.8 μL DE-PPD, 46.8 μL of tick/tick cell samples or XA/KA standards, 9.37 μL POD, 9.37 μL of 30 mM hydrogen peroxide were added and mixed well. The mixture was allowed to stand at room temperature for 3 min. For blank solution, 9.37 μL water was used instead of hydrogen peroxide. Absorbance of the reaction mixtures was measured within 5 min at 750 nm.
7
Quantitative Analysis of Neurochemicals
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The analysis procedure
followed that described in the literature.19 (link) In brief, the hippocampus tissue samples were homogenized using
deionized water. We then transferred the supernatant to the following
analysis. The Agilent 6470 triple quadrupole liquid chromatography–mass
spectrometer/mass spectrometer system was used. The chromatographic
separation was achieved on an ACQUITY UPLC HSS T3 Column, 100 Å,
1.8 μm, 2.1 × 100 mm (Waters Co., Milford, MA, US) at 40
°C. The mobile phase consisted of (A) water with 0.1% formic
acid (FA) and (B) methanol with 0.1% FA at a flow rate of 0.4 mL/min.
The gradient elution was programmed as follows: 0–8 min, 50%
A; 8–8.1 min, 50–0% A; 8.1–10 min, 0% A; 10–10.1
min, 0–100% A; 10.1–12 min, 100% A.
The mass spectrometric
detection was performed using multiple reaction monitoring with an
electrospray ionization source in positive mode. Ion source parameters
were optimized with the isocratic mobile phase composition without
column separation. The conditions were as follows: ion spray voltage,
5000 V; temperature, 300 °C; Sheath gas flow, 11 psi; nebulizer
gas, 45 psi; and heater gas, 40 psi. Data acquisition and processing
were performed with the Agilent MassHunter Workstation Software. The
standard solutions of 5-HIAA, tryptophan, kynurenine, kynurenic acid
(KA), and 3-hydroxykynurenine (3-HK) were purchased from Sigma-Aldrich
(Burlington, MA, US).
followed that described in the literature.19 (link) In brief, the hippocampus tissue samples were homogenized using
deionized water. We then transferred the supernatant to the following
analysis. The Agilent 6470 triple quadrupole liquid chromatography–mass
spectrometer/mass spectrometer system was used. The chromatographic
separation was achieved on an ACQUITY UPLC HSS T3 Column, 100 Å,
1.8 μm, 2.1 × 100 mm (Waters Co., Milford, MA, US) at 40
°C. The mobile phase consisted of (A) water with 0.1% formic
acid (FA) and (B) methanol with 0.1% FA at a flow rate of 0.4 mL/min.
The gradient elution was programmed as follows: 0–8 min, 50%
A; 8–8.1 min, 50–0% A; 8.1–10 min, 0% A; 10–10.1
min, 0–100% A; 10.1–12 min, 100% A.
The mass spectrometric
detection was performed using multiple reaction monitoring with an
electrospray ionization source in positive mode. Ion source parameters
were optimized with the isocratic mobile phase composition without
column separation. The conditions were as follows: ion spray voltage,
5000 V; temperature, 300 °C; Sheath gas flow, 11 psi; nebulizer
gas, 45 psi; and heater gas, 40 psi. Data acquisition and processing
were performed with the Agilent MassHunter Workstation Software. The
standard solutions of 5-HIAA, tryptophan, kynurenine, kynurenic acid
(KA), and 3-hydroxykynurenine (3-HK) were purchased from Sigma-Aldrich
(Burlington, MA, US).
8
Selective Receptor Blockade in Neurotransmission
9
Metabolite Profiling of Biological Samples
10
Quantification of Kynurenine Pathway Metabolites
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PIA (2-pyridinecarboxylic acid), DPIA (2,6-pyridinediarboxylic acid), quinolinic
acid (QIA: 2,3-pyridinedicarboxylic acid), quinaldinic acid (QA), kynurenic acid
(KA), xanthurenic acid (XA), and kynurenine (KY) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Zinc acetate and other chemicals were
purchased from Fujifilm-Wako Pure Chemical (Osaka, Japan). All the chemicals
were of analytical reagent grade, and used without further purification. The
handling of serum was approved by Teikyo University Ethical Review Board for
Medical and Health Research Involving Human Subjects (Ethical committee approval
No. 21-104 and approval list of 25 February 2011). Blood was collected from 1
healthy subject and serum was prepared at 4°C. Human serum was stored at −30°C
until analyzed. PIA in frozen serum is stable for at least a week.
acid (QIA: 2,3-pyridinedicarboxylic acid), quinaldinic acid (QA), kynurenic acid
(KA), xanthurenic acid (XA), and kynurenine (KY) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Zinc acetate and other chemicals were
purchased from Fujifilm-Wako Pure Chemical (Osaka, Japan). All the chemicals
were of analytical reagent grade, and used without further purification. The
handling of serum was approved by Teikyo University Ethical Review Board for
Medical and Health Research Involving Human Subjects (Ethical committee approval
No. 21-104 and approval list of 25 February 2011). Blood was collected from 1
healthy subject and serum was prepared at 4°C. Human serum was stored at −30°C
until analyzed. PIA in frozen serum is stable for at least a week.
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