Remifentanil (batch number: 120801, Ren Fu Co, China) and sevoflurane (batch number: 08100931, Heng Rui Co., China) were supplied by the Department of Anesthesiology of Drum Tower Hospital (Nanjing, China). Remifentanil (80 µg/kg) was dissolved in saline (NaCl 0.9%) and infused subcutaneously over a period of 30 min (rate, 0.8 mL/h) using an apparatus pump. Control animals (sham-operated rats) underwent a sham procedure that consisted of the administration of sevoflurane plus the same volume of saline in identical conditions. BDNF-sequester TrkB/Fc (Sigma, USA) of 5 µg was dissolved in 10 µL saline. PNU-120596 (Sigma Chemical Co., St. Louis, MI), an α7-nAChRs agonist, was dissolved in 5% DMSO. The intrathecal injection of TrkB/Fc (5 µg) or PNU-120596 (8 µg) was performed 15 min before remifentanil was subcutaneously infused. A KCC2 inhibitor VU0240551 (Sigma, USA) was dissolved in 10 µL 0.01% DMSO.9 (link),31 (link) An intrathecal dose of 0.8 µg VU0240551 was administered 15 min before surgery in rats given PNU-120596. The intrathecal injections were made into the subarachnoid space on the midline between the L5 and L6 vertebrae in unanesthetized mice, according to the method of Hylden and Wilcox.32 (link) A Hamilton microsyringe with a 30-gauge needle was used.
Pnu 120596
Manufactured by Merck Group
Sourced in United States
PNU-120596 is a selective positive allosteric modulator (PAM) of the alpha7 nicotinic acetylcholine receptor (α7 nAChR). It is used in research applications to potentiate the activity of the α7 nAChR, which is involved in various physiological processes.
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Lab products found in correlation
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Choline chloride, by Merck Group (1 mentions)
Memantine, by Merck Group (1 mentions)
Memantine, by Bio-Techne (1 mentions)
Methyllycaconitine, by Merck Group (1 mentions)
Pnu 120596, by Bio-Techne (1 mentions)
Lps of escherichia coli, by Merck Group (1 mentions)
Solutol, by Merck Group (1 mentions)
Probenecid, by Thermo Fisher Scientific (1 mentions)
Trypsin, by PanEco (1 mentions)
Acetylthiocholine iodide, by Merck Group (1 mentions)
Chaps, by Merck Group (1 mentions)
Amphotericin b, by PanEco (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Scp0139, by Merck Group (1 mentions)
Ac devd afc, by Bio-Techne (1 mentions)
Ac lehd afc, by Bio-Techne (1 mentions)
Equine serum bche, by Merck Group (1 mentions)
Hepes, by PanEco (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
6 protocols using pnu 120596
1
Modulation of Remifentanil-Induced Hyperalgesia
2
Choline Chloride and PNU-120596 Administration
3
Investigating α7 nAChR Modulation
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LPS of Escherichia coli (serotype 0127: B8) and methyllycaconitine (MLA), an α7 nAChR antagonist, were purchased from Sigma-Aldrich (St. Louis, MO, USA). PNU120596 and memantine were purchased from Tocris Bioscience (Ellisville, MO, USA). MLA and memantine were dissolved in normal saline (0.9% NaCl), whereas PNU120596 was reconstituted in saline containing 5% dimethyl sulfoxide and 5% Solutol (Sigma, St. Louis, MO, USA). All drugs were injected intraperitoneally in a volume of 10 ml/kg of body weight.
4
Adolescent ABAE and PNU-120596 Effects
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Adolescent male (n = 39) and female (n = 41) Wistar rats were used in the experiment. The experimental design was a 2 × 3 between subject protocol. Rats received low-dose ABAE treatment (2 g/kg, gavage) or water which began on PND28 and were simultaneously administered PNU-120596 (0, 3, or 5 mg/kg; Sigma-Aldrich, Inc, St. Louis, MO, USA). ABAE treatment was intermittent (4 days of the week) until PND48. ABAE exposed to 2 g/kg EtOH should have resulted in a peak blood ethanol concentration (BEC) of approximately 35–40 mg% (Vetreno et al., 2020 (link)). The primary reason why both sexes were not tested in the two adult conditions was primarily financial. These prolonged experiments are extremely costly. The same real life constraints (and the general unwillingness for funding agencies to support research preventing the negative consequences of ABAE) limited the dose of EtOH exposure during adolescence and dose of PNU tested. Individuals reading scientific manuscripts must ask the following questions; could they obtain funding for the ideal research project they demand from a publication, and if they were a reviewer for a grant application would they support funding for a full parametric analysis?
5
Neuronal Cell Culture Reagents
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l -Glutamine, fetal bovine serum, penicillin, streptomycin, amphotericin B, HEPES, Hanks’ salts solution, trypsin, DMEM, (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from PanEco, Moscow, Russia. HEPES, KCl, CaCl2, MgCl2, DMSO, Triton X-100, d -glucose, non-essential amino acids, Hoechst 33258, human erythrocyte AChE, equine serum BChE, acetylthiocholine iodide, CHAPS, EDTA, dithiothreitol, PNU 120596, protease inhibitor cocktail, SCP0139, and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) were from Sigma-Aldrich, St. Louis, MO USA. Arachidonic acid and Z-VAD-FMK were purchased from Cayman Europe, Hamburg, Germany. Ac-DEVD-AFC and Ac-LEHD-AFC were from Tocris Bioscience, Bristol, UK. Fluo-4AM and probenecid were from Thermo Fisher Scientific, Waltham, MA, USA.
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6
Tat-Induced Neurotoxicity Assay with Pharmacological Modulation
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The Tat-containing and control neurobasal conditioned medium was similarly prepared except for use of an increased number of seeding cells, 48 h culturing after transfection, change to neurobasal medium with 1% GlutaMAXTM, then 24 h after the medium change and saved as Tat-containing neurobasal conditioned medium (Tat-CM) or control neurobasal conditioned medium (Ctrl-CM). To treat primary neurons and primary cortical neuron-astrocyte co-cultures, 98% Tat-containing or control neurobasal conditioned medium and 2% B27 (50×) were used to replace the 50% culture medium. To activate α7 nAChR in cell cultures, 1 μM PNU-120596 accompanied with 0.5 μM PNU-282987 (Sigma-Aldrich, Cat no. P6499) (PAM+P2) was applied into the conditioned medium. Both of them were dissolved in DMSO at high concentrations (PNU-120596: 100 mM, PNU-282987: 50 mM) as stocks. A Nikon Eclipse TE2000-S microscope with a 10× objective was used to capture the images of microglia in the bright field, and the images were converted using the Ilastik program to visualize the cell morphology, and then were quantitated by the Cellprofiler program. Three images were randomly captured from each well.
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