We purchased normal human epidermal keratinocytes (NHEKs) from EpiLife (Cascade Biologics, Portland, OR, USA). Cells were cultured in basal keratinocyte growth medium (EpiLife), supplemented with human keratinocyte growth supplement (HKGS) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) in a 5% CO2 incubator. Passages 2~9 were used for all experiments. NHEKs (1×104 cells/well) were seeded into 96 well plates. After application of capsaicin and capsazepine (Sigma-Aldrich, St. Louis, MO, USA) at various concentrations, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) assay according to the manufacturer’s instructions. NHEKs at 70% confluence were stimulated with capsaicin (Sigma-Aldrich). The expression of TRPV1, LL37, KLK-5, TNF-α, VEGF, IL-1α, IL-1β, IL-8, and PAR2 was evaluated at times ranging from 0 to 12 h and at various concentrations of capsaicin (0.1, 1, 5, 10, and 20 µM). Regulation of these mediators induced by capsaicin (20 µM) was also evaluated after the application of a specific TRPV1 antagonist, capsazepine (Sigma-Aldrich) at various concentrations (1, 5, 10, and 20 µM). capsazepine was added 30 min before capsaicin stimulation and incubated for the indicated times.
Capsazepine
Manufactured by Merck Group
Sourced in United States, Germany, France, Sao Tome and Principe
Capsazepine is a synthetic compound primarily used as a laboratory reagent. It functions as a competitive antagonist of the capsaicin receptor, also known as the transient receptor potential cation channel subfamily V member 1 (TRPV1). Capsazepine is utilized in research settings to study the role of TRPV1 in various physiological and pathological processes.
Automatically generated - may contain errors
Lab products found in correlation
Capsaicin, by Merck Group (48 mentions)
Hc 030031, by Merck Group (8 mentions)
Indomethacin, by Merck Group (6 mentions)
Ruthenium red, by Merck Group (6 mentions)
Acetylsalicylic acid, by Merck Group (5 mentions)
Formalin, by Merck Group (5 mentions)
Glibenclamide, by Merck Group (4 mentions)
Menthol, by Merck Group (4 mentions)
Glutamate, by Merck Group (4 mentions)
Naloxone, by Merck Group (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
L glutamic acid, by Merck Group (3 mentions)
Sb366791, by Merck Group (3 mentions)
Orthovanadate, by Merck Group (3 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Merck Group (3 mentions)
Triton x 100, by Merck Group (3 mentions)
N acetyl l cysteine, by Merck Group (3 mentions)
Morphine, by Merck Group (3 mentions)
Acetic acid, by Merck Group (3 mentions)
90 protocols using capsazepine
1
Evaluating Capsaicin's Effects on NHEKs
2
Ion Channel Blocker Assay Protocol
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The ion channel blockers were purchased as follows: verapamil (Merck, V4629), mibefradil (Merck, M5441), glybenclamide (Merck, G0639), 4-aminopyridine (Merck, 275875), L-cis diltiazem (Merck, D2521), bupivacaine (Merck, B5274), DCPIB (Tocris, Bio-Techne R & D Systems, S.LU, Madrid, Spain,1540), capsazepine (Merck, C191), gadolinium (Tocris, 4741) and GsMTx-4 (Smartox Biotechnology, Saint-Egrève, France, 08GSM001). The antibodies used are listed in Supplementary Table S1 . All other reagents were purchased from Merck unless indicated otherwise.
3
Modulation of Hydrogen Sulfide Signaling
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siRNA for CBS (hydroxylamine), CSE (dl -propargylglycine), TRPV1, TRPV3, TRPV6, and TRPM4 were purchased from Santa Cruz. H2S donor NaHS was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Capsaicin and capsazepine were purchased from EMD Millipore (Billerica, MA, USA). The concentrations of Capsaicin and capsazepine used for the treatment were 1 μM.
4
Terpene and Capsaicin Compound Synthesis
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General chemicals were from VWR (West Chester, PA) and Sigma Aldrich (St. Louis, MO). Terpenes, beta-Myrcene, Capsaicin, and Capsazepine were from Sigma Aldrich.
5
Transepithelial Transport Assay Using Ussing Chambers
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Transwell-grown cell monolayers were regarded as suitable for the transepithelial measurements when the net TEER values exceeded 300 Ω . cm 2 . Confluent cell monolayers were mounted into modified Ussing chambers. Krebs-Ringer-Buffer pH 7.4 was added to both compartments of the chamber (composition of KRB: 116.4 mM NaCl, 5.4 mM KCl, 0.78 mM NaH 2 PO 4 , 25 mM NaHCO 3 , 5.55 mM glucose, 15 mM HEPES, 1.8 mM CaCl 2 , 0.81 mM MgSO 4 ). Sodium-free solutions contained equivalent amounts of KCl, KH 2 PO 4 and KHCO 3 , respectively, whereas in Cl --free solutions, equivalent amounts of the relevant gluconate salts were used.
Chambers and bathing solutions were maintained at 37°C and continuously stirred. The transepithelial potential difference (PD) was clamped to 0.0 mV using a standard fourelectrode voltage clamp (DVC-1000, World Precision Instruments, Sarasota, FL), and the short-circuit current (I SC ) was continuously recorded using Chart5 for Windows (ADInstruments, Oxfordshire, UK). Voltage impulses of 0.5 mV were given every 50 s in order to monitor the TEER of cell monolayers. Cell monolayers were allowed to reach steady state basal I SC before the compound of interest was added. The used stock solutions were 50 mM Evans blue, 5 mM capsazepine, 10 mM icilin, 2.5 mM CFTR(inh) 172, 50 mM amiloride and 100 mM ouabain (all in DMSO; all from Sigma-Aldrich).
Chambers and bathing solutions were maintained at 37°C and continuously stirred. The transepithelial potential difference (PD) was clamped to 0.0 mV using a standard fourelectrode voltage clamp (DVC-1000, World Precision Instruments, Sarasota, FL), and the short-circuit current (I SC ) was continuously recorded using Chart5 for Windows (ADInstruments, Oxfordshire, UK). Voltage impulses of 0.5 mV were given every 50 s in order to monitor the TEER of cell monolayers. Cell monolayers were allowed to reach steady state basal I SC before the compound of interest was added. The used stock solutions were 50 mM Evans blue, 5 mM capsazepine, 10 mM icilin, 2.5 mM CFTR(inh) 172, 50 mM amiloride and 100 mM ouabain (all in DMSO; all from Sigma-Aldrich).
6
Krebs-Henseleit Physiological Salt Solution Protocol
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The physiologic salt solution used was Krebs-Henseleit, which had the following
composition: 118.0 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl2, 1.2
mmol/L MgSO4, 25.0 mmol/L NaHCO3, 1.2 mmol/L
KH2PO4, 10.0 mmol/L glucose). Solutions with high KCl
content involved addition of appropriate amounts of a 3-M KCl solution (in distilled
water) directly to the organ bath to achieve the desired concentration. For some
experiments, barium ions (Ba2+) substituted for Ca2+ in the
physiologic salt solution.
(±)-β-Citronellol (95% purity; Code C83201), ACh (PubChem ID 24891113), atropine (ID
24890401), 5-hydroxytryptamine (ID 24278124), L-NG-nitroarginine methyl
ester (L-NAME; ID 24278011), tetraethylammonium (TEA; ID 24277874), sodium
orthovanadate (ID 24899708), capsazepine (ID 24277967), indomethacin (INDO; ID
24278173), A-967079 (CAS Number 1170613-55-4), HC-030031 (CAS Number 349085-38-7),
thapsigargin (ID 24278762) and verapamil (ID 24277881) were purchased from
Sigma-Aldrich (USA).
In general, stock solutions were prepared in distilled water and stored at -20°C.
β-Citronellol was dissolved directly in physiologic solution containing 2% Tween 80
and sonicated immediately before addition in the bath chamber. The maximum
concentration of the vehicle in the organ bath was 0.01% (v/v).
Salts (all of analytical grade) were purchased from Sigma-Aldrich or Merck
(Germany).
composition: 118.0 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl2, 1.2
mmol/L MgSO4, 25.0 mmol/L NaHCO3, 1.2 mmol/L
KH2PO4, 10.0 mmol/L glucose). Solutions with high KCl
content involved addition of appropriate amounts of a 3-M KCl solution (in distilled
water) directly to the organ bath to achieve the desired concentration. For some
experiments, barium ions (Ba2+) substituted for Ca2+ in the
physiologic salt solution.
(±)-β-Citronellol (95% purity; Code C83201), ACh (PubChem ID 24891113), atropine (ID
24890401), 5-hydroxytryptamine (ID 24278124), L-NG-nitroarginine methyl
ester (L-NAME; ID 24278011), tetraethylammonium (TEA; ID 24277874), sodium
orthovanadate (ID 24899708), capsazepine (ID 24277967), indomethacin (INDO; ID
24278173), A-967079 (CAS Number 1170613-55-4), HC-030031 (CAS Number 349085-38-7),
thapsigargin (ID 24278762) and verapamil (ID 24277881) were purchased from
Sigma-Aldrich (USA).
In general, stock solutions were prepared in distilled water and stored at -20°C.
β-Citronellol was dissolved directly in physiologic solution containing 2% Tween 80
and sonicated immediately before addition in the bath chamber. The maximum
concentration of the vehicle in the organ bath was 0.01% (v/v).
Salts (all of analytical grade) were purchased from Sigma-Aldrich or Merck
(Germany).
7
Endocannabinoid Regulation of hBM-MSCs Adipogenesis
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hBM-MSCs were purchased from Lonza, Inc. (Walkersville, MD, USA). hBM-MSCs were cultured according to the manufacturer’s instructions with minor modifications. hBM-MSCs were maintained in Dulbecco’s modified eagle’s medium (DMEM) with low glucose (1 g/L glucose) containing 10% fetal bovine serum (FBS) (Lonza), supplemented with antibiotics and GlutamaxTM (Invitrogen, Carlsbad, CA, USA). To induce adipogenesis, hBM-MSCs were cultured to 100% confluence. Three days after the confluence, the medium was exchanged with DMEM with high glucose (4.5 g/L glucose) supplemented with 10% FBS, 10 μg/ml insulin, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (IDX medium). After inducing adipogenesis in hBM-MSCs, IDX media were exchanged at every 72 hours. For the treatment of endocannabinoids, AEA, 2-AG and NADA were freshly added to the IDX medium during the medium exchange. AEA, 2-AG, NADA, capsazepine, rimonabant, glibenclamide, aspirin and troglitazone were purchased from Sigma-Aldrich (Sigma Aldrich, St. Louis, MO, USA).
8
Pharmacological Evaluation of Nociception
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The following drugs were used: (i) acetylsalicylic acid (ASA), apamin, atropine, bradykinin, caffeine, capsaicin, capsazepine (CAPZ), charybdotoxin, glibenclamide, haloperidol, l -glutamic acid, phorbol 12-myristate 13-acetate (PMA), pindolol, tetraethylammonium chloride, and yohimbine were purchased from Sigma-Aldrich (St. Louis, MO, USA); (ii) naltrindole hydrochloride, nor-binaltorphimine dihydrochloride and β-funaltrexamine hydrochloride were purchased from Tocris Bioscience (Ellisville, Missouri, USA); and (iii) acetic acid, dimethyl sulfoxide (DMSO), and methanol were purchased from Fisher Scientific (England). bradykinin, capsaicin, l -glutamic acid, and PMA were dissolved in physiological saline (0.9% (w/v) NaCl), while ASA, MECN, and CAPZ were dissolved in distilled water containing 10% DMSO (v/v). The vehicle used alone had no effects per se on the nociceptive responses in mice. All drugs, chemicals, and MECN solutions were administered in 10 mL/kg volumes and were freshly prepared just before being used.
9
Investigating Menthol and Formaldehyde Effects on Neurons
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After allowing the neurites to freely extend for 48 hours, the DRG neurons were pretreated with capsazepine (a TRPV1 antagonist, 10 μM) (Sigma-Aldrich) for 30 minutes. Thereafter, the neurons were treated and cultured for 24 hours as follows: (1) formaldehyde group (n = 5), DRG neurons were exposed to formaldehyde (Sigma-Aldrich) 10 μM; (2) menthol group (n = 5), DRG neurons were exposed to menthol (Sigma-Aldrich) 300 μM; (3) PD98059 + formaldehyde group (n = 5), PD98059 (an ERK1/2 inhibitor, 10 μM) (Cell Signaling Technology, Danvers, MA, USA) was added 30 minutes before formaldehyde (10 μM) exposure; (4) PD98059 + menthol group (n = 5), PD98059 (10 μM) was added 30 minutes prior to menthol (300 μM) exposure; (5) control group (n = 5), DRG neurons treated only with the TRPV1 receptor antagonist capsazepine (10 μM). Because TRPA1 and TRPV1 channels interact in neurons (Ruparel et al., 2011), capsazepine, a TRPV1 antagonist, was used to block the effects of TRPV1.
10
Pharmacological Reagent Preparation
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Nicotine and capsazepine were obtained from Sigma (St Louis, MO, USA), AM251 (1-[2,4-dichlorophenyl]-5-[4-iodophenyl]-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide) and AM630 ([6-iodo-2-methyl-1-[2-(4-morpholinyl-) ethyl]-1H-indol-3-yl] (4-methoxyphenyl) methanone) were obtained from Tocris (Ellisville, MO, USA).
AM251, AM630, and capsazepine were dissolved in DMSO. Stock solutions of the other drugs were dissolved in distilled water. Solutions were stored at −20 °C until use. The drugs were diluted in distilled water to the required final concentration on the day of use.
AM251, AM630, and capsazepine were dissolved in DMSO. Stock solutions of the other drugs were dissolved in distilled water. Solutions were stored at −20 °C until use. The drugs were diluted in distilled water to the required final concentration on the day of use.
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