8-Methoxypsoralen (8-MOP) (Sigma-Aldrich) was added to the cell culture medium at 25 ng/ml for 4 h. Cells were irradiated at a dose 6 J/cm2 in DMEM without phenol red and FBS containing 8-MOP using a Bio-Sun irradiation apparatus (Vilbert Lourmat, Marne-la-Vallee, France) with maximum emission at 365 nm in the UVA spectral region (340–450 nm). Following irradiation, the medium was replaced with fresh medium, which was changed twice a week thereafter. AT-Ex treatment was added at 2% immediately following PUVA and refreshed at each medium change. PUVA-treated control cells were cultured with 2% FBS. Experiments were stopped 2 weeks after PUVA treatment since at this time point senescent biomarkers are stably expressed.
8 methoxypsoralen 8 mop
Manufactured by Merck Group
Sourced in United States
8-methoxypsoralen (8-MOP) is a naturally occurring compound that belongs to the class of psoralens. It is a photosensitizing agent with the core function of facilitating photochemical reactions in various applications.
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Lab products found in correlation
Anti mouse igg, by Rockland Immunochemicals (1 mentions)
Erk1 2, by Santa Cruz Biotechnology (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Lapatinib gw 572016, by LC Laboratories (1 mentions)
Bt474 skbr3, by American Type Culture Collection (1 mentions)
Mcf 7 t47d, by American Type Culture Collection (1 mentions)
Hff cell line, by American Type Culture Collection (1 mentions)
Arbutin, by Merck Group (1 mentions)
Thiazolyl blue tetrazolium bromide, by Merck Group (1 mentions)
2 2 azobis 2 amidinopropane dihydrochloride, by Merck Group (1 mentions)
Hydroquinone, by Merck Group (1 mentions)
Anti phospho p38, by Bioworld Technology (1 mentions)
Anti p38, by Bioworld Technology (1 mentions)
O tetradecanoyl phorbol 13 acetate, by Merck Group (1 mentions)
Anti tyrp 2, by Bioworld Technology (1 mentions)
Anti mitf, by Bioworld Technology (1 mentions)
Anti tyrosinase, by Bioworld Technology (1 mentions)
L dopa, by Merck Group (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
10 protocols using 8 methoxypsoralen 8 mop
1
Photochemotherapy for Cellular Senescence
2
Inducing Senescence in Human Dermal Fibroblasts
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A primary culture of human dermal fibroblasts (HDFs) was prepared and maintained in our laboratory in DMEM (high-glucose) supplemented with 10% FBS. The number of population doublings (PDs) of HDFs was calculated based on the equation PDs log(A/BC)/log2, where A, B, and C indicate the number of collected cells, the number of plated cells and the attachment efficiency, respectively. The doubling time of HDF was measured with PDs, and the young cells used in this study represent cells with doubling times of approximately 24 h. The doubling time of replicative senescent fibroblasts was 14 days. For the UVA-induced senescent fibroblasts, HDF samples were washed once with PBS and placed in fresh PBS. The cells were treated with 8-methoxy-psoralen (8-MOP; Sigma-Aldrich) at 25 ng/mL for 16 h and irradiated with UVA (wavelength 320-400 nm, maximum peak 350 nm) using a LZC-1 photoreactor system (Luzchem Research Inc. Ontario, Canada). Several doses of UVA were tested according to methods described in previous reports 13 (link), 14 (link), and a dose of 5 J/cm2 was chosen for the experiment because it maintained the fibroblasts in a fully senescent state in which more than 75% show SA-β-Gal staining without cell death. Sham-irradiated HDF was rinsed and placed into an irradiator box without UV irradiation. After irradiation, the cells were maintained in DMEM for 7 days.
3
Establishing Lapatinib-Resistant Breast Cancer Cell Lines
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ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA). Lapatinib resistant breast cancer cells (rBT474; rSKBR3) were generated and maintained in culture as previously described [16] (link)–[18] (link). Cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum and L-glutamine from GIBCO (Grand Island, NY) growing in a humidified atmosphere of 5% CO2 at 37°C. IRDye 800 conjugated affinity purified anti-rabbit IgG and anti-mouse IgG were from Rockland (Gilbertsville, PA). Alexa Fluor 680 goat anti-rabbit IgG were purchased from Molecular Probes (Eugene, OR). Anti-PARP (Poly (ADP-ribose) Polymerase) monoclonal antibody was from BD PharMingen (San Jose, CA). 8-Methoxypsoralen (8MOP) and the 4G10 anti-phosphotyrosine (p-tyr) antibody were purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal antibodies to c-ErbB2 and EGFR were purchased from Neo Markers (Union City, CA). PARP cleavage product was obtained from Cell Signaling (Beverly, MA). Antibodies to Akt1/2, phospho-Akt1/2 (S473), phospho-Akt1/2 (T308), phospho-Erk1/2 and Erk1/2 were purchased from Santa Cruz (Santa Cruz, CA). Lapatinib (GW572016) and neratinib (HKI-272) [19] were purchased from LC Laboratories (Woburn, CA).
4
Melanogenesis Regulation in Vitro
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Arbutin, 3-isobutyl-1-methylxanthine (IBMX), L-DOPA (L-3,4-dihydroxyphenylalanine), sodium hydroxide, thiazolyl blue tetrazolium bromide (MTT), O-tetradecanoyl phorbol-13-acetate (TPA), 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH), 8-methoxypsoralen (8-MOP), 3-Isobutyl-1-methyl-2,6(1H,3H)-purinedione (IBMX), and hydroquinone were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). All other reagents and chemicals were high-grade and commercially available. Antibodies bought from Bioworld Technology (St. Louis Park, MN, USA) included anti-Tyrosinase, anti-TYRP-1, anti-TYRP-2, anti-MITF, anti-phospho-p38, and anti-p38, anti-phospho-ERK, and anti-ERK, anti-phospho-JNK, and anti-JNK.
5
Cigarette Smoke-Induced Apoptosis Regulation
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The cigarettes used in the study were Kentucky reference cigarettes 3R4F from the University of Kentucky (Lexington, KY, USA), containing 9.4 mg of tar and 0.726 mg of nicotine per cigarette; (−) nicotine standard was obtained from Accustandard (New Haven, CT, USA). HPLC-grade methanol, formic acid (FA), and ammonium acetate were purchased from Merck (Darmstadt, Germany); 8-methoxypsoralen (8-MOP) dissolved in dimethyl sulfoxide (DMSO) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from GIBCO-BRL (Grand Island, NY, USA). Antibodies specific to caspase-3, C-caspase-3, Poly (Adenosine Diphosphate–Ribose) Polymerase (PARP), Cleaved Poly (Adenosine Diphosphate–Ribose) Polymerase (C-PARP), Bcl-2 Associated X Protein (Bax) and B cell lymphoma-2 (Bcl-2) were purchased from Cell Signaling Technology (Danvers, MA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody, penicillin, streptomycin, and cell counting kit 8 (CCK-8) were purchased from Beyotime (Shanghai, China).
6
Inhibiting DNA Repair Pathways
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Oxaliplatin and cisplatin (Sigma-Aldrich) were dissolved in PBS, aliquoted and stored at -20°C. Treatment concentration and duration are indicated for each experiment. 8-methoxypsoralen (8-MOP, Sigma-Aldrich) dissolved in DMSO was used in 50 μM concentration for 2 h. To inhibit PARP, cells were treated for 24 h with 10 μM olaparib dissolved in PBS (Selleckchem). To inhibit POLB, cells were treated for 24 h with 500 μM pamoic acid (Sigma-Aldrich) dissolved in 200 mM NaCl and 20 mM Tris–HCl. To inhibit transcription, cells were treated for 1 h with 1 μM flavopiridol (Sigma-Aldrich), dissolved in DMSO. Trolox (Sigma-Aldrich) was dissolved in ethanol and applied for 24 h prior to an experiment at a concentration of 600 μM.
7
Isolation and Culture of Human Immune Cells
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Human peripheral blood was from anonymous healthy donors at the Blood Bank (Oslo University Hospital, Norway). The human peripheral blood mononuclear cells (PBMC) were grown in RPMI-1640 growth medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. A human T cell lymphoma cell line (Jurkat) was also cultured in the same medium. 5-Aminolevulinic acid (ALA) (Sigma Aldrich, St Louis, MO, USA) was dissolved in PBS to a concentration of 1 M and further diluted. The stock solution of 1 M 8-methoxypsoralen (8-MOP) (Sigma Aldrich) was prepared in chloroform and further diluted. The stock solution of 0.1 mg/mL phytohemagglutinin (PHA) was prepared in PBS. All the stocks mentioned above were stored at −20 °C until further use.
8
Phototherapy Protocol for Mouse Models
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PUVA or NB-UVB therapy was given every second day at a dose of 1500 mJ/cm2 (PUVA) or 200 mJ/cm2 (NB-UVB), starting at day 1 using Waldmann UVA 236 equipment (Waldmann GmbH, Villingen-Schwenningen, Germany). In the case of PUVA, mice were painted on their backs with 200 microliter 8-methoxypsoralen (8-MOP) (Sigma-Aldrich, St. Louis, MO) in ethanol (at a concentration of 0.1 mg/ml), as previously described (59 (link), 60 (link)). The mice were then kept for 15 min in individual compartments of standard cages to allow penetration of 8-MOP before UVA irradiation.
9
Psoralen-Based Pathogen Reduction
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Amotosalen HCl 3mM solution was obtained from the Cerus INTERCEPT Blood System for Platelets Pathogen Reduction System Dual Storage Processing Set and stored in light-protected aliquots at 4C. 4'-aminomethyltrioxsalen hydrochloride (AMT) was from Cayman Chemical (Ann Arbor, MI); 8-methoxypsoralen (8-MOP) was from Sigma-Aldrich (St Louis, MO). 8-MOP and AMT were dissolved in DMSO and stored as aliquots at -80C prior to use.
10
Photochemical and Pharmaceutical Compounds
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8-Methoxypsoralen (8-MOP) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anthracene, indomethacin, ketoprofen, naproxen, nifedipine, nimodipine, nitrendipine, promethazine, piroxicam, quinine, retinol, tamoxifen, pirfenidone, acetone, and sterilized 0.5 w/v% methyl cellulose (MC) solution were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Acridin was from Cayman Chemical Company (Ann Arbor, MI, USA). Amlodipine and benzoyl peroxide were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Bufexamac, chlorpromazine, diclofenac, furosemide, gatifloxacin, and nalidixic acid were obtained from LKT Laboratories, Inc. (St Paul, MN, USA). Haloperidol was purchased from MP Biomedicals LLC (Santa Ana, CA, USA). Ibuprofen was from Polysciences Inc. (Warrington, PA, USA). Amiodarone, lomefloxacin, and sparfloxacin were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Dimethyl sulfoxide (DMSO) was from Dojindo Laboratories (Kumamoto, Japan).
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