Fraxetin
Manufactured by Merck Group
Sourced in United States, Germany
Fraxetin is a laboratory equipment product manufactured by Merck Group. It is a chemical compound used in various analytical and research applications. Fraxetin is a crystalline solid with a specific molecular structure. Its core function is to serve as a reagent or intermediate in scientific experiments and procedures. No further details on intended use or interpretation are provided.
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Lab products found in correlation
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Hplc grade solvent, by Merck Group (1 mentions)
Catechin, by Phytolab (1 mentions)
Gallic acid, by Phytolab (1 mentions)
Tyrosol, by Phytolab (1 mentions)
Hydroxytyrosol, by Phytolab (1 mentions)
Oleuropein, by Phytolab (1 mentions)
Procyanidin b1, by Phytolab (1 mentions)
Fraxetin, by Phytolab (1 mentions)
Verbascoside, by Phytolab (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Esculetin, by Fujifilm (1 mentions)
Coumarin, by Nacalai Tesque (1 mentions)
Osthenol, by ChemFaces (1 mentions)
Bergamottin, by ChromaDex (1 mentions)
Esculin, by Merck Group (1 mentions)
11 protocols using fraxetin
1
Antibody and MAPK Inhibitors Analysis
2
Bioactive Compounds Sourcing and Analysis
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Hydroxytyrosol, tyrosol, gallic acid, fraxetin, oleuropein, verbascoside, catechin, luteolin 3,7 glucoside, procyanidin B1, quercitin 3-O-glucoside, and elenoic acid were obtained from PhytoLab GmbH & Co. (Vestenbergsgreuth Germany). fraxetin was from Sigma Chemical Co (St Louis, MO). Fetal bovine serum (FBS) was from Hyclone Perbio Sciences (Helsingborg, Skane Lan, Sweden). Human interleukin (IL)-1β, IL-6 and IL-8 enzyme-linked immunosorbent assay (ELISA) Kits II were from BD Biosciences Pharmingen (San Jose, CA, USA). HPLC-grade solvents and all other reagents and chemicals were from Sigma Chemical Co (St. Louis, MO, USA).
3
MTT Assay for Cell Proliferation
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Cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after cells were treated with fraxetin (both from Sigma, St. Louis, MO, USA). MCF-7 cells were seeded in 96-well plates at a concentration of 1×105/ml and each well contained 100 µl. After 24 h, fraxetin was added to 10, 20, 40 and 60 µM final concentrations. The control group was treated without fraxetin. The cells were incubated for 24 and 48 h at 37°C in 5% CO2 and then culture medium was changed. MTT (10 µl) was added to each well at a final concentration of 5 mg/ml. After incubating for 4 h, the optical density (OD) at 570 nm was measured by a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The inhibition rate was calculated as: Inhibition rate (%) = (OD value of control group - OD value of experimental group/OD value of control group) × 100%.
4
Antimicrobial Effects of Coumarins
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The antimicrobial activity of scopoletin and fraxetin (Sigma Aldrich) against single bacterial strains was assayed in liquid culture in 50% tryptic soy broth (TSB, 15g/L; Sigma Aldrich). scopoletin and fraxetin stocks were prepared in sterile DMSO (Sigma Aldrich) and stored at -80°C. Bacterial colonies were picked from TSA plates into liquid TSB and grown for 2–3 days at 25°C with 180 rpm agitation. Liquid cultures were subcultured by diluting 1:100 into fresh TSB and incubated for 1–2 h. In a clear flat-bottom 96-well microtiter plate, 100 μl of subculture were added to 100 μl of fresh TSB media supplemented with scopoletin or fraxetin for a final 50 μM concentration, or equivalent DMSO negative control. The microtiter plate was sealed with a clear adhesive film to prevent evaporation. Growth was monitored kinetically in a microplate reader (Infinite M200 PRO, Tecan) with 30 seconds of shaking followed by measurement of optical density (OD) at 600 nm in four locations per well every 30–60 min for 18–20 h. The OD in each experiment was expressed as the average of triplicate wells per condition. Relative growth (Figure 2 D) was calculated by dividing the average final OD measurement of each strain and indicated condition by the average OD in the coumarin-free control.
5
Synthesis and Characterization of Coumarin Compounds
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Standard compounds of coumarins were purchased as follows: coumarin (Nacalai), 7-methoxycoumarin (Tokyo Chemical Industry, Tokyo, Japan), 4-methoxycoumarin (Wako Pure Chemicals, Osaka, Japan), 6-methoxycoumarin (Wako), umbelliferone (Wako), daphnetin (Wako), esculetin (Wako), fraxetin (Sigma), bergaptol (Extrasynthese, Geray, France), bergamottin (ChromaDex, Irvine, CA, USA), osthenol (ChemFaces, Wuhan, China), and geraniol (Kanto Chemical Industry, Tokyo, Japan).
6
Quantitative Metabolite Analysis via LC-MS
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All buffer constituents and solvents were of reagent or HPLC grade and obtained from either Roth (Karlsruhe, Germany), Sigma-Aldrich (St Louis, MO, USA) or J. T. Baker (Deventer, The Netherlands). Coumarin standards were purchased from PhytoLab (Vestenbergsgreuth, Germany; scopolin, isofraxidin, fraxin), and Sigma-Aldrich (esculin, esculetin, fraxetin, 4-methyl-umbelliferon, scopoletin). [2,2,4,4-2H]citric acid and [2,3,3-2H]malic acid were from CDN isotopes (Point Claire, QC, Canada), [2,2,3,3-2H]succinic acid and methoxyamine HCl from Sigma-Aldrich, and Silyl-991 from Macherey-Nagel (Düren, Germany).
7
Assessing Coumarins' Impact on Bacterial Growth
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WCS417 and its Tn5-mutant derivative Mob10 [42 (link)] were transferred, using sterile wooden toothpicks, from KB to Cook’s cytophaga semi-solidified medium (CCM; 0.2% tryptone and 0.3% agar in distilled water) amended with 0, 0.25, 0.5, 1 or 2 mM of scopoletin, fraxetin, or esculetin (Sigma-Aldrich, Inc., St. Louis, MO, USA). Appropriate amounts of the respective coumarins were dissolved in 80% methanol prior to dilution in CCM. The final concentration of methanol was corrected to 3.2% in all plates, including control plates without any coumarin. Plates were sealed with Parafilm and incubated at 28 °C for 70 h. Colony surface areas on 4 replicate plates was subsequently measured using Image J (version 1.53e). Analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) were performed using the stats package for R.
8
Spectrophotometric Iron(II) Assay Protocols
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Esculetin
(6,7-dihydroxycoumarin, C9H6O4, >98%,
Alfa Aesar), scopoletin (6-methylesculetin,
C10H8O4, >99%, Sigma-Aldrich),
fraxetin
(7,8-dihydroxy-6-methoxycoumarin, C10H8O5, >98%, Sigma-Aldrich), iron(II) chloride tetrahydrate
(FeCl2·4H2O, >99%, Sigma-Aldrich), iron(III)
chloride
hexahydrate (FeCl3·6H2O, >99%, Merck),
Ferrozine (C20H13N4NaO6S2·xH2O, >98%, ACROS
organics), ammonium acetate (CH3COONH4, >98%,
Merck), sodium hydroxide (NaOH, >99%, Merck), hydrochloric acid
(HCl,
30% supra pure grade, Merck), and sodium chloride (NaCl, >99.5%,
Merck)
were used as received without further purification. Piperazine-1,4-bis(propane-sulfonic
acid) (PIPPS, C10H22N2O6S2, pKa1 = 3.73, pKa2 = 7.96, >97% pure, Merck), 2-morpholinoethanesulfonic
acid monohydrate (MES, C6H14N2·H2O, pKa = 6.06, >99%, Merck),
3-(N-morpholino)propanesulfonic acid (MOPS, C7H15NO4S, pKa = 7.20,
>99%, Carl Roth GmbH + Co), 1,4-dimethylpiperazine (DEPP, C6H14N2, pKa1 = 4.48,
pKa2 = 8.58, >98% Sigma-Aldrich), and N,N,N′,N′-tetramethylethylenediamine (TEEN, (CH3)2NCH2CH2N(CH3)2, pKa1 = 6.58, pKa2 = 9.88, >99.5%, Sigma-Aldrich) were used as pH buffers.
All experimental and analytical solutions were prepared with ultrapure
water (resistivity > 18.2 MΩ·cm, TOC < 2 ppb, Milli-Q,
Millipore).
(6,7-dihydroxycoumarin, C9H6O4, >98%,
Alfa Aesar), scopoletin (6-methylesculetin,
C10H8O4, >99%, Sigma-Aldrich),
fraxetin
(7,8-dihydroxy-6-methoxycoumarin, C10H8O5, >98%, Sigma-Aldrich), iron(II) chloride tetrahydrate
(FeCl2·4H2O, >99%, Sigma-Aldrich), iron(III)
chloride
hexahydrate (FeCl3·6H2O, >99%, Merck),
Ferrozine (C20H13N4NaO6S2·xH2O, >98%, ACROS
organics), ammonium acetate (CH3COONH4, >98%,
Merck), sodium hydroxide (NaOH, >99%, Merck), hydrochloric acid
(HCl,
30% supra pure grade, Merck), and sodium chloride (NaCl, >99.5%,
Merck)
were used as received without further purification. Piperazine-1,4-bis(propane-sulfonic
acid) (PIPPS, C10H22N2O6S2, pKa1 = 3.73, pKa2 = 7.96, >97% pure, Merck), 2-morpholinoethanesulfonic
acid monohydrate (MES, C6H14N2·H2O, pKa = 6.06, >99%, Merck),
3-(N-morpholino)propanesulfonic acid (MOPS, C7H15NO4S, pKa = 7.20,
>99%, Carl Roth GmbH + Co), 1,4-dimethylpiperazine (DEPP, C6H14N2, pKa1 = 4.48,
pKa2 = 8.58, >98% Sigma-Aldrich), and N,N,N′,N′-tetramethylethylenediamine (TEEN, (CH3)2NCH2CH2N(CH3)2, pKa1 = 6.58, pKa2 = 9.88, >99.5%, Sigma-Aldrich) were used as pH buffers.
All experimental and analytical solutions were prepared with ultrapure
water (resistivity > 18.2 MΩ·cm, TOC < 2 ppb, Milli-Q,
Millipore).
9
Macrophage Differentiation and Signaling Pathways
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Mouse macrophage RAW264.7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Fetal bovine serum (FBS) was obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The Cell Counting Kit (CCK)-8 was purchased from Dojindo Molecular Technologies, Inc., (Kumamoto, Japan). Alpha-modified Eagle’s medium (α-MEM) was obtained from Gibco; Thermo Fisher Scientific, Inc. Fraxetin, glycine, Tris, sodium dodecyl sulfate (SDS), NaCl, Acid Phosphatase kit and other reagents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). M-CSF and RANKL were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). The SYBR Premix Ex Taq II and Prime Script RT reagent kits were purchased from Takara Bio, Inc. (Otsu, Japan). Antibodies against phosphorylated (p-) JNK1/2 (cat. no. 9251), total JNK1/2 (cat. no. 9252), p-p38 (cat. no. 9211), total p38 (cat. no. 8690), p-ERK1/2 (cat. no. 9101), total ERK1/2 (cat. no. 9102), MMK6 (cat. no. 9264) and β-actin (cat. no. 4967) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA) and the working dilution was 1:1,000 for all.
10
Extraction and Characterization of Bioactive Compounds
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Fraxetin (7,8-dihydroxy-6-methoxy-coumarin), quinizarin (1,4-dihydroxy-anthraquinone), emodin (1,3,8-trihydroxy-6-methyl-9,10-anthracenedione), lapachol (2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone) were purchased from Sigma-Aldrich. Vismione H and madagascine were obtained from PGE2 fraction of Psorospermum glaberimum as previously described [6 ]. Other chemicals were from the usual commercial sources with the highest purity available.
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