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Cp99994

Manufactured by Bio-Techne
Sourced in United Kingdom

CP99994 is a laboratory equipment product manufactured by Bio-Techne. This device is designed to perform specific functions within a laboratory setting. The core function of this product is to facilitate certain processes or procedures required in a research or analytical environment. However, a detailed and unbiased description of the specific capabilities and intended use of this equipment cannot be provided without the risk of extrapolation or interpretation.

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5 protocols using cp99994

1

AITC and Capsaicin Sensitivity Assay

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AITC (Sigma) was dissolved in dimethyl sulfoxide (DMSO) (Sigma) to obtain 10 mM stock solution. Further dilutions were made with ECS solution to reach final concentrations of 100 μM. Capsaicin (Sigma) was dissolved in DMSO to obtain a 10 mM stock solution. Further dilutions were made with ECS or Hank’s solution to reach final concentrations of 330 or 100 nM, respectively. Penicillin-streptomycin was purchased from Gibco. D-MEM-low glucose, collagenase type XI, deoxyribonuclease I, horse serum, newborn calf serum, fetal bovine serum, poly-D-lysine, glycine, NGF, pertussis toxin (PTX), SP, HK-1 were purchased from Sigma. CP99994, AMG 9810 and HC 030031 were purchased from Tocris.
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2

Chemical Reagents and Compounds

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Atropine was purchased from Wako Pure Chemical (Osaka, Japan). Carbachol (CCh) and pyr3
were purchased from Sigma-Aldrich (St. Louis, MO, USA). CP99994 and GR159897 were
purchased from Tocris Bioscience (Bristol, UK). Neurokinin (NK) A and substance P (SP)
were purchased from Peptide Institute (Osaka, Japan).
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3

Tethered Ligand Induced Signaling in HEK293 Cells

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Tethered ligand induced signaling was assessed in HEK293 cells 24 hours after transfection. For soluble and lipidated peptides, 20 hours following transfection, cells were stimulated in serum-free medium for an additional 4 hours. The activity of all experimental ligands (MTLs and SMALs) was compared to a 4 hour treatment of receptors with amidated endogenous ligands: s-SubP-NH2 for NK1-3R, s-CCK4-NH2 for CCK2R, and sulfated s-CCK8-NH2 for CCK1R. For antagonist assays, CP 99994 or YM022 (Tocris, Minneapolis, MN) were added concurrently with soluble agonist for 4 hours. With tethered ligands, antagonists were added 4 hours after transfection; activity was assessed following an additional 20 hour incubation. Quantification of luciferase and β-galactosidase activities were performed as previously described [6] (link). Data were analyzed by nonlinear curve fitting using Graph Pad Prism 5.0 software.
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4

Substance P Modulation of Pain Responses

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Evans blue dye (EBD; 50 mg/ml in saline; Sigma-Aldrich, USA), the SP receptor agonist substance P acetate salt hydrate (SP; 0.5 mg/ml in saline; 30 μL/loci, subcutaneous (s.c.); Sigma-Aldrich), the SP receptor antagonist (+)-(2S, 3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine (CP-99994; 33 mmol/ml in saline; 30 μL/loci, s.c.; TOCRIS, UK), were used (McLean et al., 1993 ). The transient receptor potential channel and vanilloid subfamily member 1 (TRPV1) agonist capsaicin (0.05%; 20 μL/loci, s.c.; Sigma-Aldrich), was dissolved in vehicle consisting of 10% alcohol and 10% Tween 80 in saline.
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5

Modulation of Stress Responses in Mice

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C57Bl/6 mice were treated intraperitoneally (i.p.) with the NK1 receptor antagonist CP99994 (25-50 mg/kg, Tocris; dissolved in saline) 30 minutes prior to measurements. As a negative control, one group of animals received the same amount of saline. As a positive control in acute stress tests, one animal group was treated intraperitoneally (i.p.) with citalopram hydrobromide, a selective serotonin reuptake inhibitor (10 mg/kg, Tocris; dissolved in saline) 30 minutes prior to measurements.
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