Tissue SNS activity was assessed by measuring DOPA accumulation after inhibiting catecholamine synthesis by blocking L-aromatic amino acid decarboxylase with NSD1015 (100 mg/kg, Sigma) as described previously [25] (link). To study SNS response to cold, mice were placed at 6 °C for 30 min prior to injection of saline or NSD and kept at 6 °C for another 2 h before tissues were collected. Tissue DOPA content was quantified by reverse phase liquid chromatography.
Nsd1015
Manufactured by Merck Group
NSD1015 is a laboratory instrument developed by Merck Group. It is designed to perform precise measurements and analyses in a research or laboratory setting. The core function of NSD1015 is to provide accurate and reliable data for scientific investigations.
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Lab products found in correlation
Vasopressin, by Bio-Techne (2 mentions)
L phenyalanine, by Merck Group (2 mentions)
Sb206553, by Bio-Techne (2 mentions)
Gr127935, by Bio-Techne (2 mentions)
Rx821002, by Bio-Techne (2 mentions)
Cytometric bead array kit, by BD (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Fluoxetine, by Merck Group (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Tnf α, by BD (1 mentions)
Ifn γ, by BD (1 mentions)
Il 17, by BD (1 mentions)
Il 17, by R&D Systems (1 mentions)
Kynurenine, by Merck Group (1 mentions)
5 protocols using nsd1015
1
Measuring Sympathetic Nervous System Activity
2
Cytokine Profiling in Autoimmune Models
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Serum samples were collected from CIA mice at day 36 post-immunization and the concentrations of IL-6, IL-17, TNFα and IFNγ were measured by cytometric bead array kits (BD Biosciences). In other experiments, splenocytes from DBA/1 mice were treated with 5-HTP in the presence of kynurenine (20 μg/ml, Sigma-Aldrich), fluoxetine (5 μg/ml, Sigma-Aldrich) or NSD-1015 (8.4 μg/ml, Sigma-Aldrich) and stimulated with anti-CD3 plus anti-CD28 Abs for 36 hours. The levels of TNFα (eBioscience, San Diego, CA, USA), IL-17 (R&D Systems, Minneapolis, MN, USA), IFNγ (eBioscience) and IL-2 (eBioscience) in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA).
3
Detailed ACSF compositions for in vitro experiments
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Two kinds of ACSF were used in in vitro experiments: mACSF in the dissection dish before recording and nACSF in the recording chamber. mACSF was composed of (in mM) 118 NaCl, 24 NaHCO3, 1.5 CaCl2, 3 KCl, 5 MgCl2, 1.4 NaH2PO4, 1.3 MgSO4, 25 d -glucose, and 1 kynurenic acid. nACSF was composed of (in mM) 122 NaCl, 25 NaHCO3, 2.5 CaCl2, 3 KCl, 1 MgCl2, 0.5 NaH2PO4, and 12 d -glucose. Both types of ACSF were saturated with carbogen (95% O2-5% CO2) and maintained at pH 7.4. The drugs added to the nACSF were 5-HTP, clorgyline, pargyline, tryptamine, L-tryptophan, tyramine, L-tyrosine, 2-phenylethylamine, L-phenyalanine, NSD1015 (Sigma-Aldrich), α-methyl-5-HT, octopamine, SB206553, RX821002, GR127935, dobutamine, vasopressin (Tocris) and zolmitriptan (AstraZenica). All drugs were first dissolved as a 10 – 50 mM stock solution in water before final dilution in ACSF for in vitro ventral root reflexes recording and DIC microscopy or in saline for in vivo oxygen measurements and two photon microscopy, with the exception of zolmitriptan, which was dissolved in minimal amounts of DMSO (DMSO final concentration in nACSF was 0.04%). DMSO alone had no effect on in vitro LLR in vehicle controls, as compare to nACSF control state.
4
Detailed ACSF compositions for in vitro experiments
5
Quantifying L-DOPA in Striatal Slices
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Dorsal striatal slices were incubated for 5 min with T1AM (10 μM) or tyramine (100 μM), and then for 15 min with T1AM or tyramine along with the L -amino acid decarboxylase inhibitor NSD-1015 (100 μM, Sigma-Aldrich). After the removal of the solutions, tissue slices were frozen and sonicated (10,000 g for 10 min) in 100 μL perchloric acid (0.1 mM). The pellets were resuspended in 100 μl 1% sodium dodecyl sulfate and the protein content was determined. The level of L -DOPA in the supernatant was determined using HPLC coupled to an electrochemical detection system with a refrigerated microsampling unit (model CMA/200; CMA Microdialysis, Kista, Sweden). The HPLC apparatus comprised an HPLC pump (model 2150; Pharmacia LKB Biotechnology AB, Uppsala, Sweden) that kept a constant flow of 0.2 mL/min of the mobile phase (0.12 m NaH2PO4H2O; 0.09 m EDTA, 0.05 mm 1-octanesulfonic acid, and 15% methanol, pH 4.2) and a pressure of ∼0.5 bar on a reverse-phase ion pair C-18 column prepacked with Biophase ODS 5 μm particles (BAS, West Lafayette, IN, United States). L -DOPA was detected with an amperometric detector (model LC-4C; BAS) and a glassy carbon electrode set at 0.75 V. The limit of detection was ∼10 nM.
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