Primary human astrocytes (catalog #: 1800, ScienCell, Carlsbad, CA, USA) were grown to ∼80% confluency and infected with HIV-1SF162 (p24 = 1 ng/mL; from Dr. Jay Levy through the NIH AIDS Research and Reference Reagent Program; Germantown, MD, USA) followed by exposure with 10 μM each of e mtricitabine, r itonavir, a tazanavir (ERA) or l opinavir, a bacavir, r altegravir (LAR) alone or in combination with 500 nM of morphine sulfate (Sigma-Aldrich, St. Louis, MO, USA) for 3, 5, and 7 days. HIV infection was quantified by p24 levels using a p24 antigen ELISA (ZeptoMetrix, Buffalo, NY, USA) and by RT-PCR using the long terminal repeat (LTR) primers (F-3): 5'-TTTGTTATATTTTGTGAGTTTGTAT-3' (nucleotide position: 200 to 224, 9285 to 9309), reverse primer (R-1), 5'-CAAAAAACTCCCAAACTCAAATCTA-3' (nucleotide position: 496 to 472, 9581 to 9557). Concentration of the antiretroviral drugs are based on previously published literature by others (Cohen et al., 2017 (link); Ene et al., 2011 (link); Kravcik et al., 1999 (link); Nooka and Ghorpade, 2017 (link)) and cell viability assay. Morphine at a concentration of 500 nM has been previously shown to fully activate the u-opioid receptor as well as to enhance HIV mediated neurotoxicity (El-Hage et al., 2005 (link); Gurwell et al., 2001 (link); Hauser et al., 1996 (link); Stiene-Martin and Hauser, 1991 (link)).
Morphine sulfate
Manufactured by Merck Group
Sourced in United States, Germany
Morphine sulfate is a laboratory chemical used in analytical and research applications. It is a salt of the organic compound morphine, which is the primary active alkaloid found in opium. Morphine sulfate is commonly used as a reference standard and in the development of analytical methods for the identification and quantification of morphine and related compounds.
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Lab products found in correlation
Naloxone, by Merck Group (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Ketamine, by Merck Group (3 mentions)
Oxycodone hydrochloride, by Merck Group (3 mentions)
Menthol, by Merck Group (2 mentions)
Naloxone, by Santa Cruz Biotechnology (2 mentions)
Gf109203x, by Bio-Techne (2 mentions)
Y 27632, by Merck Group (2 mentions)
Damgo, by Merck Group (2 mentions)
Formalin, by Merck Group (2 mentions)
Capsaicin, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Naltrexone, by Merck Group (2 mentions)
Naloxone hydrochloride, by Merck Group (2 mentions)
Methadone hydrochloride, by Merck Group (2 mentions)
Indomethacin, by Merck Group (2 mentions)
Tween 20, by Merck Group (2 mentions)
P24 antigen elisa, by ZeptoMetrix (1 mentions)
Primary human astrocytes, by ScienCell (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
55 protocols using morphine sulfate
1
HIV Infection of Primary Astrocytes
2
Analytical Reagents and Assay Kits
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In the present study, all reagents were of analytical grade. As an extraction solvent, 99% absolute ethanol was used (Exodus Scientific, São Paulo, SP, Brazil). Ultrapure water, acetonitrile, and concentrated formic acid solution, obtained from Merck (Darmstadt, Germany), were used to prepare the mobile phase and solubilize the sample for LC-MS analyses. The dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA), ALT, AST, urea, and creatinine assay kits was from VIDA (Belo Horizonte, MG, Brazil). The acetic acid was from (ACA) and formaldehyde from (Vetec Química Fina, Rio de Janeiro, RJ, Brazil), the indomethacin from (Sigma-Aldrich, St. Louis, MO, USA) and morphine sulfate from (Cristália, Rio de Janeiro, RJ, Brazil).
3
Modulation of Pain Signaling Pathways
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Menthol, DAMGO, morphine sulfate, phorbol 12-myristate 13-acetate (PMA) and Y27632 were obtained from Sigma-Aldrich. The PKC inhibitor GF109203X, and TRPM8 antagonist AMTB (N-(3-Aminopropyl)-2-[(3-methylphenyl) methoxy]-N-(2-thienylmethyl) benzamide hydrochloride) and H89 were purchased from Tocris Bioscience. The specific PKCβ blocker Enzastaurin (LY317615) was purchased from Selleckchem and Naloxone from Santa Cruz Biotechnology.
4
Neuronal Autophagy Regulation in HIV-Morphine Coexposure
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Primary human neurons were purchased from ScienCell Research Laboratories (catalog number 1520) and cultured according to the manufacturer’s instructions. Cells were treated with supernatants from uninfected or HIV-1-infected (30 pg/mL HIV-1 p24) microglia and 500 nM morphine sulfate (Sigma-Aldrich; St. Louis, MO, USA; catalog number M8777) alone or in combination for 24 h and fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked in 10% milk/0.1% goat serum, and immunolabeled. Primary antibodies were anti-p62/SQSTM1 (Novus Biologicals; catalog number NBP1-48320) and anti-MAP2 (Abcam; catalog number ab5392), both used at a 1:100 dilution. Immunoreactivity was visualized with secondary antibodies from Molecular Probes conjugated to Alexa Fluor 594 dye (catalog number A11037) for p62/SQSTM1 and Alexa Fluor 488 dye (catalog number A11039) for MAP2, both used at a 1:500 dilution. DAPI staining was used to label cell nuclei.
5
Cannabinoid and Opioid Receptor Agonists
6
Solvent-Assisted Drug Preparation
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Morphine sulfate and ibuprofen sodium salt were purchased from Sigma-Aldrich (St-Louis, MO, USA). Vitamin C (acid ascorbic), sodium bicarbonate (NaHCO3) and cisplatin were purchased from Tocris (Ellisville, MO, USA). Resveratrol was provided by InvivoGen (San Diego, CA, USA). Doses of morphine and ibuprofen were selected based upon efficacy demonstrated in previous studies
[28 (link), 45 (link), 46 (link)]. Resveratrol was dissolved in 10% dimethylsulfoxide (DMSO)
[20 (link)]. Vitamin C, sodium bicarbonate (NaHCO3), morphine and ibuprofen were dissolved in normal saline (0.9% NaCl in water)
[22 (link), 28 (link), 46 (link)].
[28 (link), 45 (link), 46 (link)]. Resveratrol was dissolved in 10% dimethylsulfoxide (DMSO)
[20 (link)]. Vitamin C, sodium bicarbonate (NaHCO3), morphine and ibuprofen were dissolved in normal saline (0.9% NaCl in water)
[22 (link), 28 (link), 46 (link)].
7
Intraperitoneal Pharmacological Interventions
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Tween-20, DMSO, amitriptyline, and morphine sulfate were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Morphine was dissolved in 0.9% normal saline. All treatments were administered intraperitoneally (ip) at a volume of 10 mL/kg.
8
Subcutaneous Morphine Sulfate Injection
9
Synthesis of Azabicyclo[3.2.1]octane Derivatives
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ARN19702, ARN16186, and ARN077 were synthesized following established procedures (10 (link)). For ARN16186, sodium borohydride reduction of tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate gave an endo/exo mixture of the corresponding 3-hydroxy isomers that were separated by column chromatography. Etherification of the (exo)-3-hydroxy isomer with 4-butylphenol under Mitsunobu conditions, followed by N-Boc deprotection [trifluoroacetic acid/dichloromethane (TFA/DCM), 1:3], yielded the corresponding (endo)-3-(4-butylphenoxy)-8-azabicyclo[3.2.1]octane, which was then submitted to N-sulfonylation with 3,5-dimethyl-lH-pyrazole-4-sulfonyl chloride, in the presence of sodium hydride, to yield ARN16186. Formalin, capsaicin, morphine sulfate, gabapentin, ketamine hydrochloride (solution, 100 mg-ml−1), ketoprofen, GW7647, GW6471, and fenofibrate were from Sigma-Aldrich (St. Louis, USA). A proprietary water-soluble PEA formulation (Levagen Plus) was manufactured at Shilpa Medicare Ltd. (Raichur, India) and was a gift of Gencor (Irvine, CA). [2H4]-PEA was from Cayman Chemicals (Ann Harbor, USA). All solvents and chemicals were of the highest available grade.
10
Preparation and Use of Morphine and Related Compounds
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Morphine sulfate (Sigma M8777) was dissolved in ultrapure water, passed through 0.2 µM syringe filters into sterile Eppendorf tubes, and frozen at –20°C in the dark until use. In vitro experiments also used CTAP (Tocris 1560), naloxone (Sigma N7758), pertussis toxin (PTX; Sigma P7208), tumor necrosis factor α (TNFα; R&D systems 210-TA), ferric ammonium citrate (FAC; Sigma F5879), deferoxamine mesylate (DFO; Sigma D9533), diethylenetriaminepentaacetic acid (DTPA; Sigma D6518), and phenanthroline (Sigma 131377), which were prepared according to the manufacturer’s instructions. When neuronal cultures were appropriately aged for experiments, fresh stock solutions were diluted to working concentrations in culture medium.
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