The intracellular melanin levels were determined as previously described [19 (link),67 (link)] with some modifications. B16 cells were seeded in 6-well plates at a concentration of 2.5 × 105 cells/well and allowed to attach for 24 h. This assay was divided into 7 groups as follows: control, non-treatment; IBMX: 50 µM IBMX (PanReac AppliChem, Barcelona, Spain); theophylline: IBMX + 0.01 mg/mL theophylline (Sigma Chemical, St. Louis, MO, USA); arbutin: IBMX + 0.01 mg/mL arbutin (Sigma Chemical, St. Louis, MO, USA); PES1CMU-RBO: IBMX + 0.01 mg/mL rice bran oil; PES1CMU-DFRB: IBMX + 0.01 mg/mL de-oiled rice bran extract; and PES1CMU-H: IBMX + 0.01 mg/mL husk extract. After 48 h of incubation, cell pellets were collected and lysed with 1 N NaOH containing 10% DMSO at 80 °C for 30 min. The intracellular melanin release was measured at 405 nm using a microplate reader. The results were expressed as a fold change in melanin content compared to the control.
Arbutin
Manufactured by Merck Group
Sourced in United States, Germany, Sao Tome and Principe, Italy
Arbutin is a plant-derived compound commonly used in laboratory settings. It functions as a hydroquinone precursor, with applications in various analytical and research procedures.
Automatically generated - may contain errors
Lab products found in correlation
α msh, by Merck Group (23 mentions)
L dopa, by Merck Group (22 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (21 mentions)
Kojic acid, by Merck Group (17 mentions)
Mushroom tyrosinase, by Merck Group (14 mentions)
Quercetin, by Merck Group (11 mentions)
Caffeic acid, by Merck Group (11 mentions)
Gallic acid, by Merck Group (10 mentions)
P coumaric acid, by Merck Group (9 mentions)
Vanillin, by Merck Group (9 mentions)
L tyrosine, by Merck Group (9 mentions)
Hydroquinone, by Merck Group (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Rutin, by Merck Group (8 mentions)
Rosmarinic acid, by Merck Group (8 mentions)
Epicatechin, by Merck Group (8 mentions)
Pd98059, by Merck Group (8 mentions)
Vanillic acid, by Merck Group (7 mentions)
Sb203580, by Merck Group (7 mentions)
Sodium hydroxide (naoh), by Merck Group (7 mentions)
109 protocols using arbutin
1
Melanin Content Quantification in B16 Cells
2
Melanin Content Quantification Protocol
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The B16F10 cells (3.5×104) were cultured in 6-well plates at 37°C in an atmosphere of 5% CO2 for 24 h. After 24 h, the cells were treated with α-MSH (20 nM) and medium containing arbutin (Sigma-Aldrich; arbutin was diluted in 0.1 M phosphate buffer and used at 1 and 2 mM concentrations as a positive control) or soyasaponin Ag (25, 50 and 100 µM) for 48 h. After 48 h, the cells were washed with phosphate-buffered saline (PBS), and the cells were detached by incubation with trypsin-EDTA and centrifuged at 3,000 rpm for 5 min. The supernatant was then discarded and the cell pellet was solubilized in 1 N NaOH at 60°C for 2 h. To determine the melanin content, the absorbance was measured at 405 nm using an ELISA plate reader and then normalized to the total protein concentration.
3
Melanin Content Quantification in B16F10 Cells
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Analysis of melanin content was carried out based on modified previous reports [52 (link)]. B16F10 cells were seeded at a density of 2 × 105 in a 60 mm culture dish (SPL, New York, NY, USA). After 24 h, 200 nM α-melanocyte stimulating hormone (α-MSH; Sigma-Aldrich, St. Louis, MO, USA) and 1/100 of the total volume of maca root extracts and fermented maca root extracts or arbutin (500 µg/mL) were administered for treatment for 48 h. The culture medium was removed. Next, the cells were removed using 1 mL PBS (phosphate buffered saline) and transferred to a 1.5 mL Eppendorf tube. The collected cells were centrifuged at 13,000 rpm for 10 min. After centrifugation, the supernatant was removed, and 650 µL of 1 N NaOH (Sigma-Aldrich, St. Louis, MO, USA) solution, in which 10% DMSO (Dimethyl sulfoxide; Sigma-Aldrich, St. Louis, MO, USA) was dissolved, was added to the pellet. The mixture was dissolved at 80 °C for 1 h. Then, the tubes were vortexed and 200 µL samples were transferred to a 96-well plate and the absorbance was measured at 405 nm using a microplate reader (Thermo Fisher, Waltham, MA, USA). Moreover, 500 µg/mL arbutin (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control. The results were the averages of triplicate samples.
4
Tyrosinase Inhibition Assay with L-tyrosine
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L-tyrosine was used as a reaction substrate in the tyrosine activity assay. Briefly, 100 μl of 200 units/ml mushroom tyrosinase (Sigma-Aldrich) prepared in phosphate buffer (pH = 6.5), 50 μl of 1 mM L-tyrosinase, 10 μl of CFS from tested strains, and 40 μl of distilled water (dH2O) were added to a 96-well microplate; dH2O was used as a negative control, and 500 μM arbutin (Sigma-Aldrich) was used as a positive control. After measuring the initial absorbance at 490 nm using a microplate reader (Synergy HTX), the plate was incubated at 37°C for 30 min, and the absorbance was measured again. The cell-free tyrosinase activity was calculated using Eq. (4).
Inhibition of tyrosinase activity (%) = [(A – B) (C – D)]/ (A – B) × 100%, (4)
where A is the final absorbance of the control, B is the initial absorbance of the control, C is the final absorbance of the sample, and D is the initial absorbance of the sample.
Inhibition of tyrosinase activity (%) = [(A – B) (C – D)]/ (A – B) × 100%, (4)
where A is the final absorbance of the control, B is the initial absorbance of the control, C is the final absorbance of the sample, and D is the initial absorbance of the sample.
5
Chemoattractant-Induced Neutrophil Migration
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Arbutin, d -salicin, phenyl-β-d -glucopyranoside (PBD-Gluco), denatonium, and 5-propyl-2-thiouracil were purchased from Sigma-Aldrich (MO, USA). Recombinant CXCL2 was obtained from BioLegend (CA, USA), CXCL1 was from R&D system (MN, USA), N-formylmethionyl-leucyl-phenylalanine (fMLP) and Y-27632 were from Nacalai-Tesque (Kyoto, Japan), KD025 was from MedChemExpress (NJ, USA), and Tat-C3 was from Cytoskeleton (CO, USA). FITC-conjugated anti-Ly6G (1A8), APC/eFluor 780-anti-Ly6G (1A8), APC-anti-Ly6G (1A8), and PE-anti-CD11b (M1/70) monoclonal antibodies (mAbs) were purchased from Thermo Fisher Scientific (MA, USA). BV421-anti-CD8 (53-6.7), BV421-anti-F4/80 (BM8), Alexa Fluor 647-anti-CD4 (GK1.5), PE-anti-B220 (RA3-6B2), APC/Cy7-anti-Ly6G (1A) mAbs, and Dylight 649-anti-rabbit IgG polyclonal antibody (pAb) (Poly4064) were purchased from BioLegend. Anti-phospho-myosin light chain 2 (MLC2; Ser19) pAb was purchased from Cell Signaling Technology (MA, USA).
6
Analysis of Phytochemical Compounds from Various Sources
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The following substances and solvents were used in the study: procyanidins B1, B2, C1, A1, A2, A4, (+)-catechin, (–)-epicatechin, quercetin, quercitrin (quercetin-3-O-rhamnoside), isoquercitrin (quercetin-3-O-glucoside), avicularin (quercetin-3-O-arabinofuranoside), guaiaverin (quercetin-3-O-arabinopyranoside), rutin (quercetin-3-O-rutinose), reynoutrin (quercetin-3-O-xyloside), kaempferol, nicotiflorin (kaempferol-3-O-rutinoside), afzelin (kaempferol-3-O-rhamnoside), astragalin (kaempferol-3-O-glucoside), chlorogenic acid (3-O-caffeoylquinic acid), neochlorogenic acid (5-O-caffeoylquinic acid), cryptochlorogenic acid (4-O-caffeoylquinic acid), p-coumaric acid, arbutin, hydroquinone, α-amyrin, β-amyrin, β-sitosterol, lupeol, erythrodiol, maslinic acid, oleanolic acid, acetonitrile, acetone, and methanol, which were purchased from Sigma–Aldrich (Steinheim, Germany); hyperoside (quercetin-3-O-galactoside), procyanidin B3, uvaol, friedelin, 6″-O-acetylisoquercitrin (quercetin-3-O-(6″-acetylglucoside)), betulin, and betulinic and corosolic acids from Extrasynthese (Genay, France); ursolic acid from Carl Roth (Karlsruhe, Germany); and trifluoroacetic acid from Merck (Darmstadt, Germany). All used chemicals were of HPLC grade. Ultrapure water used in this study was prepared with Milli–Q® 180 (Millipore, Bedford, MA, USA) water purification system.
7
Melanogenesis Pathway Regulation
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α-MSH, 3-(4,5,-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), L-DOPA and arbutin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies to tyrosinase (sc-73244), TRP-1 (sc-58438), TRP-2 (sc-74439) and microphthalmia-associated transcription factor (MITF; sc-52938) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
8
Cell Viability Assay for Fibroblasts and Keratinocytes
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IHR was provided by Yunnan Hempmon Pharmaceuticals Co., Ltd. (Kunming, China). Phenol, α-naphthol, sulfuric acid, carbazole, Coomassie blue G-250, phosphoric acid, ethanol, chloroform, and monosaccharide control were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Galacturonic acid, glucuronic acid, guluronic acid, and arbutin were bought from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Human dermal fibroblast (HDF), human epidermal keratinocytes (HEK), and complete cell medium were bought from Sciencell Co., Ltd. (Carlsbad, CA, USA). 12-well plates and 96-well plates were offered by Corning Co., Ltd. (Corning, NY, USA). CCK-8 cell viability assay kit was bought from DOJINDO Biology Co., Ltd. (Tokyo, Japan) and other detection kits were offered by Takara Co., Ltd. (Takara, Japan).
9
Evaluation of Depigmenting Agents in Melanocyte Cultures
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Arbutin, kojic acid, and niacinamide were purchased from Sigma-Aldrich, dissolved in deionized water at 20 mM, and stored at −20 °C until use. At the time of use, each depigmenting agent was diluted to an appropriate concentration in a phenol red-free cell culture medium. In the case of monolayer culture, depigmenting agents were added along with α-MSH. In the case of 3D culture, the depigmenting agents were added to the cell culture medium when melanocyte aggregates were transferred to the well plate from hanging-drops at 72 hours after the cell seeding. Then the melanocyte aggregates were exposed to depigmenting agents for an additional 96 hours.
10
Melanoma Cell Signaling Modulation
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Cytochalasin D, ciliobrevin A1, α-MSH, arbutin, and kojic acid were purchased from Sigma-Aldrich (St. Louis, MO). The Smo-agonist SAG was purchased from Calbiochem (San Diego, CA). Previously validated siRNAs targeting mouse GLI2 #1 (5ʹ-CCAACCAGAACAAGCAGAACA-3ʹ ) and #2 (5ʹ-GAACACGAAGGCTGTAACAAA-3ʹ ) [25 (link)] and a negative control siRNA (5ʹ-CCUACGCCACCAAUUUCGU-3ʹ ) were synthesized by Genolution (Seoul, Korea). Melan-a and B16F1 cells cultured in 60 mm dishes were transfected with siRNA using Lipofectamine® RNAi MAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. After incubation with siRNA for 24 h, the cells were treated with each chemical for 48 h.
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