We cultured 293FT cells (Thermo Fisher Scientific) in Dulbecco’s modified Eagle’s medium (Nacalai Tesque) containing 10% fetal bovine serum at 37 °C under 5% CO2. Cells were plated at a density of 2.2 × 106 cells/100-mm dish; 24 h after plating, cells were transfected with plasmids encoding CYP2C9, CPR, and cytochrome b5 cDNA (9.6 μg, 0.2 μg, and 0.2 μg, respectively) using 30 μL of 1.0 mg/mL Polyethylenimine “Max” (Polysciences, Inc., Warrington, PA, USA), according to previously described methods [32 (link)]. After incubation for 12 h at 37 °C, 0.25 mM 5-aminolevulinic acid hydrochloride (Nacalai Tesque) and 0.25 mM iron (II) sulfate heptahydrate (FUJIFILM Wako Pure Chemical Corporation) were added to the medium. After incubation for 48 h post-transfection at 37 °C, the cells were scraped from the plates. Microsomal fractions were prepared, and protein concentrations were determined as described previously [32 (link)].
Iron 2 sulfate heptahydrate
Manufactured by Fujifilm
Sourced in Japan
Iron (II) sulfate heptahydrate is an inorganic compound with the chemical formula FeSO4·7H2O. It is a crystalline solid that appears as pale green crystals or powder. The compound is used in various laboratory and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
293ft cell, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium, by Nacalai Tesque (2 mentions)
5 aminolevulinic acid hydrochloride, by Nacalai Tesque (2 mentions)
Polyethylenimine max, by Polysciences (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Nacalai Tesque (1 mentions)
Hydrochloric acid (hcl), by Fujifilm (1 mentions)
Oleic acid, by Tokyo Chemical Industry (1 mentions)
Glutaraldehyde, by Tokyo Chemical Industry (1 mentions)
Capric acid, by Fujifilm (1 mentions)
Hanks balanced salt solution hbss, by Fujifilm (1 mentions)
Palmitic acid, by Tokyo Chemical Industry (1 mentions)
Crystal violet, by Fujifilm (1 mentions)
Calcium chloride dihydrate, by Fujifilm (1 mentions)
Myristic acid, by Tokyo Chemical Industry (1 mentions)
Stearic acid, by Tokyo Chemical Industry (1 mentions)
Potassium hydroxide, by Fujifilm (1 mentions)
Lauric acid, by Tokyo Chemical Industry (1 mentions)
Caprylic acid, by Fujifilm (1 mentions)
Linoleic acid, by Fujifilm (1 mentions)
6 protocols using iron 2 sulfate heptahydrate
1
Recombinant CYP2C9 Protein Production
2
Heterologous Expression of CYP4F2 Variants
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Wild-type and variant CYP4F2 cDNAs subcloned into pcDNA3.4 were purchased from GenScript (Piscataway, NJ, USA). Plasmids carrying CPR or cytochrome b 5 cDNAs subcloned into pcDNA3.4 were prepared as previously described (Kumondai et al., 2020) .
293FT cells (Thermo Fisher Scientific, Waltham, MA, USA) were cultured in Dulbecco's modified Eagle's medium (Nacalai Tesque) supplemented with 10% fetal bovine serum at 37 °C and 5% CO 2 . 293FT cells were plated at a density of 2.2 × 10 6 cells/100-mm dish; 24 h after plating, cells were transfected with plasmids encoding CYP4F2, CPR, and cytochrome b 5 cDNA (9.6 μg, 0.2 μg, and 0.2 μg, respectively) using 30 μL of 1.0 mg/mL Polyethylenimine "Max" (Polysciences, Inc., Warrington, PA, USA), according to previously described methods (Kumondai et al., 2020) . After incubation for 12 h at 37 °C, 0.25 mM 5-aminolevulinic acid hydrochloride (Nacalai Tesque) and 0.25 mM iron (II) sulfate heptahydrate (FUJIFILM Wako Pure Chemical Corporation) were added to the medium. After incubation for 48 h post-transfection at 37 °C, the cells were scraped from the plates. Microsomal fractions containing each CYP4F2 variant, CPR, and cytochrome b 5 were prepared as previously described (Kumondai et al., 2020) . Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific).
293FT cells (Thermo Fisher Scientific, Waltham, MA, USA) were cultured in Dulbecco's modified Eagle's medium (Nacalai Tesque) supplemented with 10% fetal bovine serum at 37 °C and 5% CO 2 . 293FT cells were plated at a density of 2.2 × 10 6 cells/100-mm dish; 24 h after plating, cells were transfected with plasmids encoding CYP4F2, CPR, and cytochrome b 5 cDNA (9.6 μg, 0.2 μg, and 0.2 μg, respectively) using 30 μL of 1.0 mg/mL Polyethylenimine "Max" (Polysciences, Inc., Warrington, PA, USA), according to previously described methods (Kumondai et al., 2020) . After incubation for 12 h at 37 °C, 0.25 mM 5-aminolevulinic acid hydrochloride (Nacalai Tesque) and 0.25 mM iron (II) sulfate heptahydrate (FUJIFILM Wako Pure Chemical Corporation) were added to the medium. After incubation for 48 h post-transfection at 37 °C, the cells were scraped from the plates. Microsomal fractions containing each CYP4F2 variant, CPR, and cytochrome b 5 were prepared as previously described (Kumondai et al., 2020) . Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific).
3
Bacterial Growth Inhibition Assay
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Lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and glutaraldehyde were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Caprylic acid, capric acid, linoleic acid, SLS, sodium chloride (NaCl), disodium hydrogen phosphate 12-water (Na2HPO4•12H2O), potassium dihydrogen phosphate (KH2PO4), sodium chloride (KCl), potassium hydroxide (KOH), hydrochloric acid (HCl), manganese (II) chloride tetrahydrate (MnCl2•4H2O), magnesium sulfate heptahydrate (MgSO4•7H2O), iron (II) sulfate heptahydrate (FeSO4•7H2O), calcium chloride dihydrate (CaCl2•2H2O), agar, crystal violet, Hank’s balanced salt solution (HBSS)(+) and 99.5% ethanol (EtOH) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). SLES was purchased from the NOF Corporation (Tokyo, Japan). The Dulbecco’s phosphate-buffered saline (d -PBS)(+) preparation reagent (with Ca and Mg) (100×) and fetal bovine serum (FBS) were purchased from Nacalai Tesque (Kyoto, Japan) and Thermo Fisher Scientific K.K. (Tokyo, Japan), respectively. WELPAS® antiseptic solution for hands (0.2%), an alcohol-based disinfectant, was purchased from Maruishi Pharmaceutical Co. Ltd. (Osaka, Japan).
4
Ozone-Induced Oxidative Probe Assay
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Reagents were obtained from the following suppliers: hydrogen peroxide 30%, iron (II) sulfate heptahydrate, NaClO, and hypoxanthine that were from Wako Pure Chemical Industries Ltd. (Osaka Japan). Xanthine oxidase was obtained from bovine milk and pyrrolidine from SIGMA-ALDRICH, Co (USA). Diethylenetriaminepentaacetic acid (DETAPAC) and NOC-7 were obtained from DOJIN MOLECULAR TECHNOLOGIES, INC. All other chemicals were obtained as reagent-grade.
Probe 1 was donated kindly from Professor K. Koide of University of Pittsburgh [6 ].
Ozone-containing water was prepared by the electrolysis of water (ozone generator: VMC JAPAN). The concentration of standard ozone-containing water was calculated from its absorbance at a wavelength of 254 nm using a molar absorptivity coefficient, ε, of 2950 mol−1Lcm−1 [7 ].
Preparation of probe 1 solution for assay: a solution of probe 1 (12.5 μmol/L) in a mixture of 5% v/v methanol in a 0.5 mmol/L phosphate buffer (pH 3.3) was prepared immediately prior to use.
Probe 1 was donated kindly from Professor K. Koide of University of Pittsburgh [6 ].
Ozone-containing water was prepared by the electrolysis of water (ozone generator: VMC JAPAN). The concentration of standard ozone-containing water was calculated from its absorbance at a wavelength of 254 nm using a molar absorptivity coefficient, ε, of 2950 mol−1Lcm−1 [7 ].
Preparation of probe 1 solution for assay: a solution of probe
5
Free Radical Scavenging Assay Protocol
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AETS, TTAU, HTAU, and cysteamine hydrochloride were kindly gifted from Sogo Pharmaceutical Co., Ltd. (Tokyo, Japan). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Labotec (Tokyo, Japan), xanthine oxidase (XOD) from bovine milk was from Sigma-Aldrich (St. Louis, MO), hypoxanthine (HX) and iron(II) sulfate heptahydrate were from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan), heparin sodium salt solution was from Mochida Pharmaceutical Co., Ltd. (Tokyo, Japan), ethylenediamine-N,N,N’,N’-tetraacetic acid, disodium salt, dihydrate (EDTA) and diethylenetriamine-N,N,N’,N’’,N’’-pentaacetic acid (DTPA) were from Dojindo (Kumamoto, Japan). Pure water was freshly prepared with a Millipore Milli-Q Labo (Bedford, MA). All other reagents were of the highest purity commercially available.
6
Extraction of DPA from B. subtilis Spores
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2,6-Pyridinedicarboxylic acid, iron(II) sulfate heptahydrate, potassium nitrate, sodium hydroxide, silver nitrate, trisodium citrate dehydrate, hydroxylammonium chloride, oxalic acid, ammonium oxalate monohydrate, and terbium (III) chloride hexahydrate were obtained from Wako Pure Chemical Industries (Japan). Branched polyethylenimine, Mw ~25,000, was obtained from Sigma Aldrich. Standard suspension of Bacillus subtilis spore (1.0 × 107 CFU/mL, Eiken Chemical Co., Ltd., Tokyo, Japan) was used as a target endospore for extraction of DPA.
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