Simultaneous profiling of multiple cytokines in addition to a set of brain-derived proteins (endocrine, neuropeptide) was performed in saliva samples in the cytokine reference laboratory located in the University of Minnesota Department of Pediatrics using a commercially available 22-plex Human Cytokine Array Panel (LUH000, R&D Systems, Minneapolis, MN, USA). Salivary levels of cytokines/chemokines, agouti-related peptide (AgRP), and prolactin were determined by multiplex method on the Luminex platform (Austin, TX, USA) with Bioplex software (BioRad, Hercules, CA, USA) using human-specific bead sets from Millipore (Billerica, MA, USA). Enzyme-linked immunosorbent assays (ELISAs) were used to determine levels of cortisol (Alpco Diagnostics), dynorphin A (Phoenix Pharmaceuticals), neuropeptide Y (RayBiotech), somatostatin (BACHEM—Peninsula Labs), and NGF (Promega). Values were interpolated from standard curves of the relevant recombinant human proteins set up on each plate. Dilution series and standard curves were run for all samples; all assays were performed in duplicate.
Dynorphin a
Manufactured by Phoenix Pharmaceuticals
Sourced in United States
Dynorphin A is a naturally occurring opioid peptide that functions as an endogenous ligand for the kappa opioid receptor. It is a component of the endogenous opioid system and plays a role in pain perception and modulation.
Automatically generated - may contain errors
Lab products found in correlation
Cortisol, by ALPCO (2 mentions)
Neuropeptide y, by RayBiotech (2 mentions)
Nerve growth factor (ngf), by Promega (1 mentions)
Bio plex software, by Bio-Rad (1 mentions)
Luminex platform, by Bio-Rad (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Perchloric acid, by Merck Group (1 mentions)
Nor binaltorphimine dihydrochloride nor bni, by Abcam (1 mentions)
2 4 6 trinitrobenzenesulfonic acid tnbs, by Merck Group (1 mentions)
Naloxone hydrochloride, by Merck Group (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
6 protocols using dynorphin a
1
Multiplex Salivary Cytokine and Neuropeptide Profiling
2
Optimized Reagents for Biomolecular Assays
3
Dynorphin A Neutralizing Antibody Protocol
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BAA and minocycline were purchased from Zelang Bio-Pharmaceutical (Nanjing, China) and Northeast Pharmaceuticals Group (Shenyang, China). Nor-binaltorphimine dihydrochloride (nor-BNI) and 5′-Guanidinonaltrindole (5′-GNTI) were obtained from Abcam (Cambridge, United Kingdom) and Sigma-Aldrich (St. Louis, MO, USA). dynorphin A (1−17) with peptide sequences of YGGFLRRIRPKLKWDNQ was synthesized by Dan Gang Peptides Co. (Hangzhou, China) with purity not less than 98%. The rabbit polyclonal antibodies neutralizing dynorphin A were purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA), with specificity to dynorphin A (100%), but not to dynorphin B (0%), β-endorphin (0%), α-neo-endorphin (0%), or leu-enkephalin (0%) according to the manufacturer’s descriptions. Its specificity was also validated by the antigen absorption test from other laboratories (Wakabayashi et al., 2010 (link); Yamada et al., 2013 (link)). All of the reagents and drugs were diluted or dissolved in 0.9% normal saline or artificial cerebrospinal fluid (ACSF).
4
Analgesic Compound Procurement and Validation
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Bulleyaconitine A (BAA), minocycline, and nor-binaltorphimine dihydrochloride (nor-BNI) were purchased from Zelang Bio-Pharmaceutical (Nanjing, China), Yuanye Biotech (Shanghai, China), and Abcam (Cambridge, United Kingdom), respectively. TNBS (2,4,6-trinitrobenzene sulfonic acid) and 5′-guanidinonaltrindole (5′-GNTI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). dynorphin A (1–17) of YGGFLRRIRPKLKWDNQ was synthesized by Dan Gang Peptides Co. (Hangzhou, China) with its purity not less than 98%. The rabbit dynorphin A antiserum was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA), with its specificity to dynorphin A (100%), but not to dynorphin B (0%), β-endorphin (0%), α-neo-endorphin (0%), or leu-enkephalin (0%), according to the manufacturer's description. Its specificity was also validated by the antigen absorption test from other laboratories [22 (link), 23 (link)]. All of the reagents and drugs were diluted or dissolved in 0.9% normal saline or artificial cerebrospinal fluid (ACSF) except for TNBS which was dissolved in the 10% ethanol/90% saline solution.
5
Pharmacological Reagents for Dynorphin Research
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BAA was purchased from Zelang Bio-Pharmaceutical (Nanjing, China) with a purity no less than 98% determined by manufacturer with high performance liquid chromatography. Morphine hydrochloride, minocycline, and pentobarbital sodium were obtained from the Northeast Pharmaceuticals Group (Shenyang, China), Yuanye Biotech (Shanghai, China), and Sinopharm Chemical Reagent Co., (Shanghai, China), respectively. Both 5′-guanidinonaltrindole (GNTI) and naloxone hydrochloride were from Sigma-Aldrich (St. Louis, MO, United States). Furthermore, the rabbit polyclonal antiserum neutralizing dynorphin A was purchased from Phoenix Pharmaceuticals (Burlingame, CA, United States). The antiserum was specific to dynorphin A (100%), but not to dynorphin B (0%), β-endorphin (0%), α-neo-endorphin (0%) or leu-enkephalin (0%) according to the manufacturer′s datasheet. Its specificity was also validated by the antigen absorption test from other laboratories (Wakabayashi et al., 2010 (link); Yamada et al., 2013 (link)). All the drugs and reagents were dissolved or diluted in 0.9% normal saline.
6
Quantification of Endogenous Peptides
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Endomorphin 2 (EM-2), Leu-enkephalin (Leu-Enk), Met-enkephalin (Met-Enk), Dynorphin A (Dyn A), Substance P (SP) and Calcitonin gene related peptide (CGRP) were purchased from Phoenix Pharmaceuticals (Belmont, CA, USA). Deuterium labeled analogue peptides were synthesized (CanPeptide, Inc., Pointe-Claire, QC, Canada) and used as internal standards.
Acetonitrile was purchased from Fisher Scientific (NJ, USA) and trifluoroacetic acid (TFA) was obtained from BDH Laboratory supplies (Poole, England, UK). Hexane and formic acid (FA) were purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Standard solutions were prepared in 0.25% TFA solution as described previously [Beaudry et al., 2009] .
Acetonitrile was purchased from Fisher Scientific (NJ, USA) and trifluoroacetic acid (TFA) was obtained from BDH Laboratory supplies (Poole, England, UK). Hexane and formic acid (FA) were purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Standard solutions were prepared in 0.25% TFA solution as described previously [Beaudry et al., 2009] .
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