2′R-ochratoxin A (2′R-OTA) was synthetized as described [23 (link)]. Ochratoxin A (OTA), human serum albumin (HSA), bovine serum albumin (BSA), porcine serum albumin (PSA), rat serum albumin (RSA), racemic warfarin, furosemide, phenylbutazone, ibuprofen, methyl orange, bilirubin, and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Fetal bovine serum (FBS), (Pan-Biotech, Aidenbach, Germany), Bioluminescent ATP Assay Kit CLSII (Roche; Basel, Switzerland), and Coomassie Brilliant Blue G-250 (Reanal; Budapest, Hungary) were used as received. Stock solutions of 2′R-OTA and OTA (both 5000 µM) were prepared in ethanol (96 v/v%, Reanal, spectroscopic grade) and stored protected from light at −20 °C.
Ochratoxin a ota
Manufactured by Merck Group
Sourced in United States
Ochratoxin A (OTA) is a mycotoxin produced by certain species of Aspergillus and Penicillium fungi. It is a common contaminant in various food and feed commodities. OTA can be detected and quantified using analytical techniques such as high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA).
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Phenylbutazone, by Merck Group (1 mentions)
Bilirubin, by Merck Group (1 mentions)
Rat serum albumin, by Merck Group (1 mentions)
Racemic warfarin, by Merck Group (1 mentions)
Porcine serum albumin, by Merck Group (1 mentions)
Furosemide, by Merck Group (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
Bioluminescent atp assay kit clsii, by Roche (1 mentions)
Coomassie brilliant blue g 250, by Reanal (1 mentions)
Dulbecco s modified eagle medium dmem, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ibuprofen, by Merck Group (1 mentions)
Methyl orange, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
Aflatoxin b1 afb1, by Merck Group (1 mentions)
Cholyl lysyl fluorescein clf, by BD (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
8 protocols using ochratoxin a ota
1
Synthesis and Evaluation of 2'R-Ochratoxin A
2
Mycotoxin Detection Using Gold Nanoparticles
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All chemicals were of analytical reagent grade and were purchased through Fisher Scientific (Waltham, MA, USA) unless otherwise noted. Pure reference standards of the three studied mycotoxins: aflatoxin B1 (AFB1), zearalenone (ZEN), and ochratoxin A (OTA) were purchased through Sigma-Aldrich (St. Louis, MO, USA) with an original concentration of 20 µg/mL, 50 µg/mL, and 50 µg/mL, respectively. Gold nanoparticles (colloidal, citrate-capped AuNPs, 50 nm at original concentration of 0.1 mg/mL) were purchased from a commercially available source (Thermo Scientific, Waltham, MA, USA).
3
Mycotoxin Exposure in Mouse Models
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Male C57Bl6/N 6–8 week-old (Janvier Labs, France) or mT/mG reporter (Muzumdar et al. 2007 (link); The Jackson Laboratory, ME, USA) mice were used in this study. The mice were housed under 12 h light/dark cycles at controlled ambient temperature of 25 °C, and were fed ad libitum with standard diet (Ssniff, Soest, Germany) and had free access to water. The experiments were approved by the local authorities. Aflatoxin B1 (AFB1) and ochratoxin A (OTA) were purchased from Sigma-Aldrich, Darmstadt, Germany (Cat. No. A6636 and O1877, respectively). OTA was also isolated from fungal cultures as described by Bittner et al. (2013 (link)) and provided by the Institute of Food Chemistry, WWU Münster, Germany. Tetramethylrhodamine–ethyl ester (TMRE) was purchased from Thermo Scientific, MA, USA (Cat. No. T669). Cholyl–lysyl–fluorescein (CLF; a bile acid analog) was obtained from BD Biosciences, California, USA (Cat. No. 451041).
4
Cytotoxicity Evaluation of Ochratoxin A
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The following is a list of all of the chemicals and their manufacturers used in this study: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) (GIBCO, ThermoFisher Scientific, Waltham, MA, USA), trypsin–EDTA (GIBCO), phosphate-buffered saline (PBS, pH = 7.2 ± 0.2) (Adwia Pharmaceuticals, El Sharkeya, Egypt), mixture of 100 IU/ml penicillin, and 100 mg/ml streptomycin, (Sigma-Aldrich, MO, USA). Ochratoxin A (OTA), catalogue number O1877, purity ≥ 98%, HPLC grade was purchased from (Sigma-Aldrich, USA), MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 0.01 M solution dimethylsulfoxide (DMSO) (ICI-UK).
5
Porcine Intestinal Cells Treated with Ochratoxin A
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The porcine intestinal cells (IPEC-J2) were cultured in media that contained 90% Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin (PS) and incubated in incubators with 5% CO2 and 37 °C. Ochratoxin A (OTA; Sigma-Aldrich, St. Louis, MO, USA) and MyD88 inhibitor (TJ-M2010-5, MedChemExpress, Monmouth Junction, NJ, USA) were diluted in dimethyl sulfoxide (DMSO), and each solution was diluted in media before treated cells. The OTA treatment protocol was the same as in our previous study, with 4 μM OTA for 48 h [27 (link)].
6
Mycotoxin Standards Purchasing Protocol
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All reagents, solvents, and mycotoxin standards (aflatoxin B1, ABF1; ochratoxin A, OTA; fumonisin B1, FB1) were purchased from Sigma-Aldrich S.r.l. (Milano, Italia).
Milli-Q quality water was produced by Millipore Progard 2 MilliQ Water Purification System. (Millipore, Milford, MA, USA).
Milli-Q quality water was produced by Millipore Progard 2 MilliQ Water Purification System. (Millipore, Milford, MA, USA).
7
Synthesis and Characterization of Ochratoxin Derivatives
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Ochratoxin A (OTA) (CAS: 303-47-9) (purity 98%) was supplied by Sigma-Aldrich (St.
Louis, MO, USA), ochratoxin alpha (OTα) (CAS: 16281-39-3) was from Romer Labs Diagnostic (Tulln, Austria). Ochratoxin B (OTB) was a gift from Fredrik C. Størmer (Norwegian Institute of Public Health). The methylated and ethylated metabolites of OTA, OTB and OTα (OTA-ethyl ester, OTA-methyl ester, OTB-ethyl ester, OTB-methyl ester, OTα-ethyl ester, OTα-methyl ester) were prepared in presence of methanol or ethanol, respectively, and a strong acid (HCl) according to Li et al. (1998) . The glutathione conjugate of OTA (OTA-GSH) was prepared according to Tozlovanu et al. (2012) (link).
Louis, MO, USA), ochratoxin alpha (OTα) (CAS: 16281-39-3) was from Romer Labs Diagnostic (Tulln, Austria). Ochratoxin B (OTB) was a gift from Fredrik C. Størmer (Norwegian Institute of Public Health). The methylated and ethylated metabolites of OTA, OTB and OTα (OTA-ethyl ester, OTA-methyl ester, OTB-ethyl ester, OTB-methyl ester, OTα-ethyl ester, OTα-methyl ester) were prepared in presence of methanol or ethanol, respectively, and a strong acid (HCl) according to Li et al. (1998) . The glutathione conjugate of OTA (OTA-GSH) was prepared according to Tozlovanu et al. (2012) (link).
8
Evaluating Compound Effects on Neural Differentiation
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The cells were exposed to the test compounds during neural commitment until the 8th day of differentiation, thus encompassing the acquisition of polarized contractile neuro-epithelial properties and the generation of neural precursors. Different concentrations of each chemical: theophylline (Sigma T1633) at 2, 6, 20, 60, 200, 600, 2000 and 6000 µM, saccharin sodium salt hydrate (Sigma S1002) at 0.002, 0.02, 0.2, 2, 20 and 50 mM, cyclopamine hydrate (CPA; Sigma C4116) at 0.016, 0.05, 0.16, 0.5, 1.6, 5 and 16 µM, valproic acid sodium salt (VPA; Sigma P4543) at 0.08, 0.25, 0.8, 2.5 and 8 mM, ochratoxin A (OTA; Sigma 32937) at 0.5, 1, 2, 4, 10, 25, 62,5 and 125 nM and mycophenolic acid (MMF; Sigma M3536) at 11.5, 23, 46, 92, 185 and 470 nM were added at day 2 and day 5 after plating. At day 8, the rosettes were counted into each well under a Leica microscope at 10x magnification and the cells then harvested. For chemicals prepared into DMSO, the final DMSO concentration was 0.1% for all conditions including untreated samples. Experiments with CPA and VPA were performed on all 4 hESC lines, except cytotoxicity testing that was done with CHES6. Experiments with OTA and MMF were performed only on CHES6. All data presented are from three independent experiments, each performed in duplicate.
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