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Nkh 477

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NKH-477 is a laboratory equipment product offered by Bio-Techne. It is a device designed for use in research and scientific applications. The core function of NKH-477 is to perform specific tasks or measurements within a controlled laboratory environment. No further details are available without the risk of providing an interpretation or extrapolation beyond the factual information.

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Lab products found in correlation

Forskolin, by Bio-Techne (3 mentions) Kt5720, by Bio-Techne (2 mentions) Menzel gl ser, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) M csf, by Thermo Fisher Scientific (1 mentions) Hepes solution, by Thermo Fisher Scientific (1 mentions) Serotonin hydrochloride, by Bio-Techne (1 mentions) Phorbol 12 myristate 13 acetate pma, by Bio-Techne (1 mentions) Ro 20 1724, by Merck Group (1 mentions) Isoproterenol hydrochloride, by Bio-Techne (1 mentions) Dantrolene sodium salt, by Bio-Techne (1 mentions) Isoproterenol hydrochloride, by R&D Systems (1 mentions) 3 isobutyl 1 methylxanthine ibmx, by MP Biomedicals (1 mentions) Ym 254890, by Focus Biomolecules (1 mentions) Fura 2 am, by Thermo Fisher Scientific (1 mentions) Complete freund s adjuvant cfa, by Merck Group (1 mentions) Matrigel, by BD (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Nerve growth factor (ngf), by Thermo Fisher Scientific (1 mentions) Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)

7 protocols using nkh 477

1

Macrophage Differentiation into Osteoclasts

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15.000 macrophages were seeded per cavity of a 24-well plate prepared with 12mm glass cover slips (Menzel-Gläser, Thermo Fisher Scientific, Braunschweig, Germany). Macrophages were cultured in osteoclast medium without neurotransmitter supply or with ACh, NA (final conc. 10−6 & 10-8M) or VIP (final conc. 10−9 & 10-11M) for 5 days. In studies involving the adenylyl cyclase activator NKH477 (cat.no. 1603, Tocris, Bristol, UK; final conc. of 10−6 and 10-8M), macrophages were differentiated for 5 days and additionally incubated in serum-free medium containing NKH477 for 24h (see section cathepsin K activity assay) and then further processed for determination of osteoclast number. Assay was performed in duplicate for each animal and condition, except for NKH477 experiments which were performed in triplicate. Afterwards, cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) using the “Acid phosphatase, Leukocyte (TRAP)” kit from Sigma-Aldrich (cat. No. 387A, Taufkirchen, Germany) following the manufacturer’s instructions. Cells containing three or more nuclei were defined as osteoclasts and were counted.
Muschter D., Schäfer N., Stangl H., Straub R.H, & Grässel S. (2015). Sympathetic Neurotransmitters Modulate Osteoclastogenesis and Osteoclast Activity in the Context of Collagen-Induced Arthritis. PLoS ONE, 10(10), e0139726.
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2

Macrophage Activation Signaling Compounds

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RPMI-1640 and penicillin-streptomyocin (P/S) from Invitrogen (Carlsbad, CA). Human AB serum and fetal bovine serum (FBS) from Mediatech (Tewksbury, MA). HEPES solution from Fisher Scientific (Fair Lawn, NJ). Dantrolene sodium salt, serotonin hydrochloride, isoproterenol hydrochloride, NKH 477, forskolin and phorbol 12-myristate 13-acetate (PMA) from Tocris Biosciences (Minneapolis, MN). isoproterenol hydrochloride from R&D Systems (Minneapolis MN). Dopamine and H89 dihydrochloride from Sigma-Aldrich (St. Louis, MO). 3-Isobutyl-1-methylxanthine (IBMX) from MP Biomedicals (Santa Ana, CA). RO-20-1724 from EMD Millipore (Temecula, CA). YM-254,890 from Focus Biomolecules (Plymouth Meeting, PA). Dopamine and the beta-adrenergic receptor agonist Isoproterenol were resuspended in distilled H2O to make stock concentrations of 10 mM, then diluted into media as needed. RO-20-1724 (200 mM), IBMX (100 mM), forskolin (10 mM), YM-254,890 (10 mM) and PMA (100 μM) were resuspended in DMSO to the indicated concentrations, then diluted into media as indicated. Human macrophage colony stimulating factor (M-CSF) was from Peprotech (Rocky Hill, NJ) and resuspended in 100 μL distilled H2O.
Nickoloff E.A., Mackie P., Runner K., Matt S.M., Khoshbouei H, & Gaskill P.J. (2019). Dopamine increases HIV entry into macrophages by increasing calcium release via an alternative signaling pathway. Brain, behavior, and immunity, 82, 239-252.
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3

Assessing Motor Function in Twitcher Mice

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Aged-matched mice from different litters received daily IP injections beginning at P10: WT mice receiving saline (n = 4); twi receiving saline (n = 3); twi receiving 1 mg/kg NKH-477 (Tocris) in saline (n = 4) were analyzed for locomotive ability and gait using the Phenoscan suite and Runwayscan software (CleverSys, Inc) at P25. Multiple parameters, including stance, stride, swing, brake, and propulsion time (milliseconds), stride length (millimeters), and average speed (millimeters/s) were collected for each animal over three compliant trials and averaged for both front and rear paws.
Folts C.J., Scott-Hewitt N., Pröschel C., Mayer-Pröschel M, & Noble M. (2016). Lysosomal Re-acidification Prevents Lysosphingolipid-Induced Lysosomal Impairment and Cellular Toxicity. PLoS Biology, 14(12), e1002583.
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4

Chemical Agents for Neuroscience Research

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ω-Agatoxin, AM-0902, AMG9810, AZ-628, 1,9-dideoxyforskolin, 6-Bnz-cAMP, 8-Br-cAMP, brain-derived neurotrophic factor (BDNF), CE3F4, 8-pCPT-2-O-Me-cAMP sodium salt (CPTOMe-cAMP), ω-conotoxin (CTX) MVIIC, [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO), ESI-05, ESI-09, diltiazem, forskolin, gabapentin, guanfacine, H89, HJC0350, ifenprodil, KT5720, L-732,138, MK-801, ML-786, NKH-477, (2R/S)-6-PNG, protein kinase inhibitor 14–22 (PKI 14–22), SNX-482, SQ22536, Torin-2, U0126 and U-73122 were from Tocris (Ellisville, MO). Baclofen and capsazepine were from Research Biochemicals International (RBI, now Sigma-Aldrich). Bovine serum albumin, cyanquixaline (6-cyano-7-nitroquinoxaline-2,3-dione) (CNQX), complete Freund’s adjuvant (CFA), ketamine, lidocaine, Phosphatase Inhibitor Cocktail 2 and common reagents were from Sigma-Aldrich. Matrigel was from BD Biosciences (San Jose, CA). Fura2-AM, nerve growth factor, neurobasal media, NuPAGE Tris-Acetate SDS gels, NuPAGE reagents and Pierce™ Protein-Free T20 (TBS) blocking buffer were from Life Technologies, Grand Island, NY. Halt™ Protease Inhibitor Cocktail and Pierce BCA Protein Assay Kit were from Thermo Fisher Scientific. Fetal bovine serum was from Irvine Scientific, Santa Ana, CA. Drugs were prepared as stock solutions of 1–10 mM in DMSO or water and then diluted in aCSF.
Chen W., McRoberts J.A., Ennes H.S, & Marvizon J.C. (2021). cAMP signaling through protein kinase A and Epac2 induces substance P release in the rat spinal cord. Neuropharmacology, 189, 108533.
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5

Synthesis of MNF Stereoisomers and Fenoterol

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(R,R’), (S,R ’), (R,S’) and (S,S’) stereoisomers of MNF as well as (R,R’)-Fenoterol (Fen) were synthesized according to previously reported synthesis scheme [1 (link), 18 (link)]. Forskolin, NKH-477, Ro 20–1724, (R)-(−)-rolipram, zardaverine, H-89 and KT 5720 were purchased from Tocris Bioscience (Ellisville, MO, USA). Epinephrine, isoproterenol, ICI-118,551 and CGP-20712A were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tritiated compounds, [3H]CGP-12177 (41.6 Ci/mmol), [3H]thymidine (10 Ci/mmol) and [35S]-protein labeling mix (0.1 Ci/mmol) were from PerkinElmer Life and Analytical Sciences (Waltham, MA, USA).
Wnorowski A., Sadowska M., Paul R.K., Singh N.S., Boguszewska-Czubara A., Jimenez L., Abdelmohsen K., Toll L., Jozwiak K., Bernier M, & Wainer I.W. (2015). Activation of β2-adrenergic receptor by (R,R’)-4’-methoxy-1-naphthylfenoterol inhibits proliferation and motility of melanoma cells. Cellular signalling, 27(5), 997-1007.
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6

Quantifying Cellular cAMP and Calcium

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For cAMP analyses, MC3T3 and PTH1R-expressing HEK cells (2.5 × 104 cells/well, 384-well plate) were treated with PTHrP peptides (1–100 nm, 15 min, n = 5/group). cAMP activity was measured with the cAMP-Glo™ Assay (Promega, Madison, WI, USA; #V1501) as per manufacturer’s instructions. The forskolin analogue NKH477 (Tocris, Bristol, UK; 10 μm, 15 min) served as a positive control. Calcium flux in response to similar concentrations of PTHrP peptides was determined using Fluo-4 Direct™ calcium reagent (Invitrogen, #F10471, 1 × 105 cells, 48-well plate). Fluorescence intensity was measured by time-lapse microscopy (EVOS FL, Waltham, MA, USA; Invitrogen) and quantified from individual cells (n = 20/group) in three fields of view per condition (Definiens, Munich, Germany).
Frieling J., Shay G., Izumi V., Aherne S., Saul R., Budzevich M., Koomen J, & Lynch C. (2017). Matrix metalloproteinase processing of PTHrP yields a selective regulator of osteogenesis, PTHrP1–17. Oncogene, 36(31), 4498-4507.
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7

Twitcher Mouse Model for Krabbe Disease

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Adult heterozygote (Galctwi/+) C57Bl/6J (B6.CE-Galctwi/J) mice were originally obtained from Dr. Ernesto Bongarzone (University of Illinois at Chicago, Chicago, IL) and used as breeder pairs to generate homozygous (twi; Galctwi/twi) twitcher mice and WT (Galc+/+) C57Bl/6J mice. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Rochester School of Medicine and Dentistry and conformed to the requirements of the Animal Welfare Act. In total, three cohorts of aged-matched mice from different litters received daily IP injections beginning at P10: WT mice receiving saline (n = 6); twi receiving saline (n = 6); twi receiving 1 mg/kg NKH-477 (Tocris) in saline (n = 8). Mice were euthanized at P35 and tissue was isolated after transcardial perfusion with 4% paraformaldehyde. Immunohistochemical analyses were completed as outlined above. For survival analysis, animals were provided moistened chow and hydragel water packs and monitored daily for weight gain. Animals were euthanized when moribund, as assessed by when the animals could no longer ambulate to maintain food and water intake or exhibited clinical signs of pain such as hunched posture and ruffled fur, as determined on a daily basis. Animals were euthanized using CO2 exposure and cervical dislocation.
Folts C.J., Scott-Hewitt N., Pröschel C., Mayer-Pröschel M, & Noble M. (2016). Lysosomal Re-acidification Prevents Lysosphingolipid-Induced Lysosomal Impairment and Cellular Toxicity. PLoS Biology, 14(12), e1002583.
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