Whole peripheral blood cultures were generated using samples collected in sodium heparin at 8 a.m., following the protocol outlined by Cassiano et al.18 (link). In brief, the blood samples were mixed with 50% (v/v) RPMI 1640 medium (Sigma-Aldrich, Saint Louis, Missouri). Two different conditions were employed: endpoint A, where the medium was supplemented with an excipient (citrate-phosphate buffer, pH 5, Merck, Darmstadt, Germany), and endpoint B, where the medium was supplemented with cyanocobalamin (Merck) at a final concentration of 1 nM. The cultures were then incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO2.
Cyanocobalamin
Manufactured by Merck Group
81 citations
Sourced in United States, Germany, United Kingdom, Australia, Brazil, Denmark
About the product
Cyanocobalamin is a form of vitamin B12, a water-soluble vitamin essential for various bodily functions. It is commonly used as a laboratory reagent for the detection and quantification of vitamin B12 levels in biological samples.
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81 protocols using «cyanocobalamin»
1
Peripheral Blood Culture Conditions
2
Spectroscopic Analysis of Cobalamin Derivatives
Hydroxocobalamin hydrochloride (HOCbl, ≥ 98%), cyanocobalamin (NCCbl, ≥ 98%), sodium cyanide (≥ 95%) and sodium chlorite (p.a., 80%; further purification as described in reference [15 (link)] did not improve this percentage) were obtained from Sigma-Aldrich (Munich, Germany) and used as received. The buffer solution used was a universal pH buffer, Britton–Robinson, it consists of a mixture of 0.04 M boric acid, 0.04 M phosphoric acid and 0.04 M acetic acid, that has been titrated to pH 7 with 0.2 M sodium hydroxide.
UV–Vis spectra were performed on a Cary 50 UV–Vis spectrophotometer (Varian, Inc., Foster City, CA, USA).
Raman spectra were measured on a Renishaw inVia Raman spectrometer coupled with a Leica microscope at 22 °C. 10 μl of each sample was dropped on a microscope slide covered with aluminum foil. The 532 nm laser line with a power of 100 mW was focused on the sample using a 5X objective. Each spectrum is represented as an average of 4 accumulations and 4 s.
NMR spectra were recorded at 20 °C unless otherwise stated, after diluting the sample (at concentrations indicated in text and Figure legends) with D2O, on a 500 MHz Bruker instrument. The solvent used was Britton–Robinson universal buffer at pH 7 prepared in D2O. A water-suppression pulse sequence was used for these measurements.
High-resolution mass spectra (HRMS) were recorded on an LTQ ORBITRAP XL mass spectrometer (ThermoScientific) using positive electrospray ionization. The instrument was externally calibrated. The samples were prepared at room temperature (22 °C) and then inserted into the instrument immediately. The following conditions were used: source voltage, 3.2 kV; sheath and auxiliary gas flow, 8 and 5 arbitrary units, respectively; vaporizer temperature 50 °C, capillary temperature 275 °C, analyzer temperature 26 °C; capillary voltage, 28 V; tube lens voltage, + 110 V. The number of microscans was set to three.
For DFT calculations, the Gaussian09 software package [21 ] was employed following the methodology previously described for Cbl complexes [17 (link)]. The Cbl models were truncated, with the lateral substituents on the corrin as well as the methyl groups on the benzimidazole replaced by hydrogen. Gas-phase geometries and frequency analyses were computed with the aid of the B3PW91 [22 (link), 23 (link)] functional at the def2-SV(P) [24 (link)] double-zeta basis set level. Long-range interactions were accounted by the use of Grimme’s D3 dispersion correction [24 (link)]. Population analyses, NMR [25 (link)] and TD-DFT derived [25 (link)] UV–Vis spectra were computed in the C-PCM solvent continuum adapted for aqueous environment [26 (link)]. In terms of methodology choice for DFT calculations, the methodology employed here was selected for its ability to best mimic trends in UV–Vis spectra as described in our previous study on hydroperoxocobalamin, thus also allowing consistency between the two sets of data [27 (link)]. DFT-derived spectral data were obtained using Chemcraft [28 ]; for the Raman simulations, 298 K and an excitation wavelength of 22,000 cm−1 were assumed.
UV–Vis spectra were performed on a Cary 50 UV–Vis spectrophotometer (Varian, Inc., Foster City, CA, USA).
Raman spectra were measured on a Renishaw inVia Raman spectrometer coupled with a Leica microscope at 22 °C. 10 μl of each sample was dropped on a microscope slide covered with aluminum foil. The 532 nm laser line with a power of 100 mW was focused on the sample using a 5X objective. Each spectrum is represented as an average of 4 accumulations and 4 s.
NMR spectra were recorded at 20 °C unless otherwise stated, after diluting the sample (at concentrations indicated in text and Figure legends) with D2O, on a 500 MHz Bruker instrument. The solvent used was Britton–Robinson universal buffer at pH 7 prepared in D2O. A water-suppression pulse sequence was used for these measurements.
High-resolution mass spectra (HRMS) were recorded on an LTQ ORBITRAP XL mass spectrometer (ThermoScientific) using positive electrospray ionization. The instrument was externally calibrated. The samples were prepared at room temperature (22 °C) and then inserted into the instrument immediately. The following conditions were used: source voltage, 3.2 kV; sheath and auxiliary gas flow, 8 and 5 arbitrary units, respectively; vaporizer temperature 50 °C, capillary temperature 275 °C, analyzer temperature 26 °C; capillary voltage, 28 V; tube lens voltage, + 110 V. The number of microscans was set to three.
For DFT calculations, the Gaussian09 software package [21 ] was employed following the methodology previously described for Cbl complexes [17 (link)]. The Cbl models were truncated, with the lateral substituents on the corrin as well as the methyl groups on the benzimidazole replaced by hydrogen. Gas-phase geometries and frequency analyses were computed with the aid of the B3PW91 [22 (link), 23 (link)] functional at the def2-SV(P) [24 (link)] double-zeta basis set level. Long-range interactions were accounted by the use of Grimme’s D3 dispersion correction [24 (link)]. Population analyses, NMR [25 (link)] and TD-DFT derived [25 (link)] UV–Vis spectra were computed in the C-PCM solvent continuum adapted for aqueous environment [26 (link)]. In terms of methodology choice for DFT calculations, the methodology employed here was selected for its ability to best mimic trends in UV–Vis spectra as described in our previous study on hydroperoxocobalamin, thus also allowing consistency between the two sets of data [27 (link)]. DFT-derived spectral data were obtained using Chemcraft [28 ]; for the Raman simulations, 298 K and an excitation wavelength of 22,000 cm−1 were assumed.
3
Cyanocobalamin Uptake Kinetics in Anabaena
For uptake measurements cultures were pre-starved for 14 days as described earlier, unless otherwise indicated. Prior to uptake experiments, Anabaena cultures were washed three times with YBG11-Co medium. Uptake was measured in 13 ml of cultures of OD750nm = 0.2. 20 µl of 57Co-cyanocobalamin (stock solution of 0.5 µCi, MP Biomedicals GmbH, Eschwege, Germany) was mixed with 13.6 µl of cyanocobalamin (stock solution 50 nM, Sigma-Aldrich, St. Louis, Missouri, USA) to adjust the final concentration to 92 pM. Cells were incubated in 50-ml falcon tubes in a water bath at 30°C under constant shaking in the dark. 0.5, 4, 8 and 16 minutes after the addition of cyanocobalamin, 2 ml of cell culture were filtered on a hydrophilic membrane (25 mm, 0.45 µm pore size, Merck, Darmstadt, Germany). After filtration, cells were washed three times with 1 ml of washing buffer (1 mM cyanocobalamin, 2 mM NaHCO3, 20 µM EDTA). Filter digestion and dissolution was previously described [64 (link)]. 10 ml of Aquasafe 300+ scintillation liquid (Zinsser Analytic, Frankfurt, Germany) was added and the 57Co-cyanocobalamin determination was done with a Hidex 300 SL liquid scintillation counter (Hidex Deutschland Vertrieb GmbH, Mainz, Germany).
Corresponding organizations : Goethe University Frankfurt, Frankfurt Institute for Advanced Studies
4
Quantification of Bioactive Compounds
Standard chemicals including gallic acid, ascorbic acid, quercetin, phloroglucinol, phylloquinone, menaquinone, and cyanocobalamin were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and solvents utilized in the study were of ACS or high-performance liquid chromatography (HPLC) grades. The commercial infant formula (Imperial Dream XO World Class 3, Namyang, Seoul, Korea) used as an analytical quality control sample was purchased from a local supermarket and stored at −70 °C.
5
Comprehensive Analytical Protocol for Fungal Beta-Glucan Quantification
All standards and reagents were purchased from Merck (UK) and were ACS grade unless otherwise stated: 4-Hydroxybenzhydrazide (PAHBAH) (H9882, Sigma Aldrich), Calcofluor White stain (18909, Sigma Aldrich), bicinchoninic acid assay kit (BCA) (BCA1), bovine serum albumin used as the BCA standard (P0914), MgSO4·7H2O (Catalogue No. M7506), Pipes buffer (Catalogue No. P6757), NH4Cl (Catalogue No. A9434), Trypticase (Catalogue No. Z699195), MnCl2·4H2O (Catalogue No. M3634), FeSO4·7H2O (Catalogue No. F7002), ZnCl2 (Catalogue No. 208086), CuCl·2H2O (Catalogue No. 224332), CoCl2·6H2O (Catalogue No. C8661), SeO2 (Catalogue No. 200107), NiCl2·6H2O (Catalogue No. 654507), Na2MoO4·2H2O (Catalogue No. M1003), NaVO3 (Catalogue No. 72060), H3BO3 (Catalogue No. B6768), acetic acid (Catalogue No. 71251), propionic acid (Catalogue No. P1386), butyric acid (Catalogue No. B103500), isobutyric acid (Catalogue No. I1754), 2-methylbutyric acid (Catalogue No. 193070), valeric acid (Catalogue No. 240370), isovaleric acid (Catalogue No. 129542), biotin (Catalogue No. B4639), folic acid (Catalogue No. F8758), calcium D-pantothenate (Catalogue No. PHR1232), nicotinamide (Catalogue No. N0636), riboflavin (Catalogue No. R9504), thiamine HCl (Catalogue No. T1270), pyridoxine HCl (Catalogue No. P6280), para-amino benzoic acid (Catalogue No. 579513), cyanocobalamin (Catalogue No. PHR1234), deuterium oxide (Catalogue No. 151882), d4-trimethylsilyl propionic acid sodium salt (Catalogue No. 269913), molecular biology grade water (Catalogue No. W4502), 5 mL Eppendorf® tubes (Catalogue No. Z768744), 1.5 mL LoBind® tubes (Catalogue No. 0030108442), paraformaldehyde (Catalogue No. P6148), MP bio fast DNA spin kit for soil (Catalogue No. 116560200, MP Biomedicals, USA), and Seward Stomacher® Classic Bags (Catalogue No. BA6041/5/500; The Laboratory Store, UK). Megazyme Ltd Mushroom and yeast beta-glucan assay kit (K-YBGL 11/19) was used for MYC, and Megazyme Ltd β-Glucan Assay Kit (Mixed Linkage) (K-BGLU 08/18) was used for OAT (Bray, Ireland).
Corresponding organizations : Quadram Institute, Norwich Research Park, Quorn (United Kingdom)
Top 5 most cited protocols using «cyanocobalamin»
1
Chronic Cobinamide and Vitamin B12 Supplementation in Mice
21 7-week-old female mice (strain 129.S6; Taconic, Denmark) were divided into three groups nd caged separately: control mice (C) n = 7 cobinamide-loaded mice (Cbi) n = 7; and vitamin B12-loaded mice (B12) n = 7. The mice had free access to water and to standard chow (Altromin maintenance diet for rats and mice (1324) (19 pmol/g B12; 0.4 pmol/g Cbi) Altromin, Germany) except during 24-h urine collection once weekly in metabolic cages. After 1 week of acclimation (age 8 weeks), an osmotic minipump (Mini-Osmotic Pump Model 2004, Alzet Cupertino, CA, USA) was implanted into each animal. The mice were anesthetised with isoflurane (IsoFlo® Vet), Abbott), and osmotic minipumps were inserted subcutaneously into the back by incision just below the neck region. After insertion, the wound was closed by absorbable suture. Prior to insertion, the pumps were equilibrated and filled following the manufacturer's instructions. The pumps were filled with either saline (0.9% NaCl) (control mice), 17 mM dicyano-cobinamide (MW: 1042.12 g/mol Sigma-Aldrich, Saint Louis, MO, USA) in 0.9% NaCl (Cbi mice), or 7 mM cyanocobalamin (MW: 1355.37 g/mol; Sigma-Aldrich) in 0.9% NaCl (B12 mice). The latter represented the maximal amount of B12 that could be dissolved. According to the manufactures, the delivery rate of the pumps is 0.25 µL/hr equivalent to delivered rate of 4.25 nmol/h for Cbi mice and 1.75 nmol/h for B12 mice. To avoid wound biting between mice, the mice were housed in individual cages for 3 days after surgery. In addition, to ameliorate pain after surgery, analgesics was put into the drinking water (buprenorphine hydrochloride 0.06 mg/ml) for 3 days post operation. 27 days after insertion of the osmotic minipumps, mice were anaesthetised using isoflurane. Following, they were sacrificed by exsanguination followed by cutting of the thorax and heart.
Corresponding organizations : Aarhus University Hospital
2
Establishing Murine Model of Vitamin B12 Deficiency
C57BL/6 mice were obtained from The Jackson Laboratory and maintained under specific pathogen–free and Helicobacter-free conditions. These mice were used to establish a murine model of VB12 deficiency by feeding them a diet depleted of any traces of VB12 (TD.180321; ENVIGO), including brewer’s yeast, which is rich in vitamins (e.g., VB12) and minerals. Mice were kept on the VB12-deficient diet for at least two generations to deplete VB12 in newborns (Ghosh et al., 2016 (link); Roman-Garcia et al., 2014 (link)). To better control nutritional intake, we compared the VB12-deficient mice that were orally gavaged with VB12 (cyanocobalamin; Sigma-Aldrich) versus PBS to elucidate VB12-dependent effects, instead of having a control group fed regular chow (Teklad global 18% protein rodent diet, catalog no. 2918; ENVIGO) that contains VB12 but also brewer’s yeast. The amount of VB12 used for rescuing VB12 deficiency was determined based on daily food consumption: one mouse typically consumes ∼5 g of regular chow containing 0.08 mg VB12/kg diet. This corresponds to a VB12 intake of ∼400 ng. Considering that breastfeeding may provide an additional source of VB12 and the limited food consumption by newborns, we designed a regimen for VB12 supplementation: 40 ng/mouse/twice a week before weaning and 400 ng/mouse/twice a week after weaning. Given that VB12-deficiency from birth can have long-lasting repercussions (Ghosh et al., 2016 (link); Roman-Garcia et al., 2014 (link)), we applied this regimen to gavage mice kept on a VB12-deficient diet for at least two generations to alleviate the effects of VB12 deficiency. Subsequently, sex- and age-matched mice from VB12 and PBS groups were used for the experiments in our studies. All experiments were conducted according to protocols approved by the Institutional Animal Care and Usage Committee of the University of Florida under protocol number 202008484 and the Department of Laboratory Animal Resources of the University of Texas Health at San Antonio under protocol numbers 20210045AB and 20210065AR.
Corresponding organizations : The University of Texas Health Science Center at San Antonio, University of Florida
3
Optimization of Bacteriocin AMA-K Production by L. plantarum
L. plantarum AMA-K was grown in 10 mL MRS broth (Biolab) for 18h at 30°C, the cells harvested by centrifugation (8000xg, 10min, 4°C), and the pellet re-suspended in 10 mL sterile peptone water. Four ml of the cell suspension was used to inoculate 200 mL of the following media: (a) MRS broth (9 ), without organic nutrients, supplemented with tryptone (20.0g/ L), meat extract (20.0g/L), yeast extract (20.0g/L), tryptone (12.5g/L) plus meat extract (7.5g/L), tryptone (12.5g/L) plus yeast extract (7.5g/L), meat extract (10.0g/L) plus yeast extract (10.0g/L), or a combination of tryptone (10.0g/L), meat extract (5.0g/L) and yeast extract (5.0g/L), respectively; (b) MRS broth, i.e. with 20.0g/L D-glucose; (c) MRS broth without D-glucose, supplemented with 20.0g/L fructose, sucrose, lactose, mannose, maltose and gluconate, respectively; (d) MRS broth with 5.0 to 50.0g/L glucose as sole carbon source; (e) MRS broth with 2.0 to 20.0g/L K2HPO4, 2.0 to 20.0g/L KH2PO4 or combination of 2.0g/L K2HPO4 and 2.0g/L KH2PO4; (f) MRS broth supplemented with 1.0 to 50.0g/L glycerol; (g) MRS broth without MgSO4; (h) MRS broth without MnSO4; (i) MRS broth without or supplemented with 5.0g/L and 10.0g/L tri-ammonium citrate; and (j) MRS broth without Tween 80 or supplemented with 0.5 to 2.0g/L.
In a separate experiment, the vitamins cyanocobalamin (Sigma, St. Louis, Mo.), L-ascorbic acid (BDH Chemicals Ltd), thiamine (Sigma) and DL-6,8-thioctic acid (Sigma) were filter-sterilised and added to MRS broth at 1.0 mg/mL (final concentration). All cultures were incubated at 30°C for 24h. Activity levels of bacteriocin AMA-K were determined as described elsewhere. All experiments were done in triplicate.
In a separate experiment, the vitamins cyanocobalamin (Sigma, St. Louis, Mo.), L-ascorbic acid (BDH Chemicals Ltd), thiamine (Sigma) and DL-6,8-thioctic acid (Sigma) were filter-sterilised and added to MRS broth at 1.0 mg/mL (final concentration). All cultures were incubated at 30°C for 24h. Activity levels of bacteriocin AMA-K were determined as described elsewhere. All experiments were done in triplicate.
Corresponding organizations : Stellenbosch University
4
Investigating Colon Microbiota Response to Vitamin B12 Levels
Brown serum flasks (20 ml, Infochroma AG, Goldau, Switzerland) containing 10 ml of sterile anaerobic Macfarlane medium adapted for batch fermentation conditions [two fold buffering capacity; pH 7.0; composition described in Bircher et al. (13 (link))] were used. The Macfarlane medium was previously developed from the analysis of intestinal content to mimic the human proximal colon chyme of Western adults (14 (link)). This medium includes the addition of a filter-sterilized vitamin solution (15 (link)), leading to a final vitamin concentration of (ng/ml medium): folic acid, 20; cyanocobalamin, 5; pyridoxine-HCl, 100; 4-aminobenzoic acid, 50; nicotinic acid, 50; biotin, 20; thiamine, 50; riboflavin, 50; phylloquinone, 0.075; menadione, 10; pantothenate, 100 (all purchased from Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). Vitamins were weighted in the dark and the vitamin solution was filtered sterilized through 0.2 μm nylon membrane filter (Infochroma AG), covered by aluminium foil and stored at 4°C before being supplemented to the autoclaved fermentation medium.
Each fecal microbial suspension was grown in three different media containing distinct levels of cyanocobalamin: (i) Control: MacFarlane medium containing normal vitamin solution (5 ng/ml cyanocobalamin); (ii) NB12: no cyanocobalamin added in the vitamin solution used for the MacFarlane medium and (iii) ExtraB12: MacFarlane medium supplemented with high dose of 2500 ng/ml cyanocobalamin final concentration. No B12 was detected in the MacFarlane medium without B12 and in Control medium by UHPLC-DAD method (measured in triplicate; detection limit 30 ng/ml). Therefore, a competitive enzyme immunoassay (RIDASCREEN FAST Vitamin B12, R-Biopharm, Switzerland) was used to quantify B12 content in MacFarlane medium, with and without vitamin mix, quantified as 8.9 ± 0.1 ng/ml and 4.1 ± 0.4 ng/ml, respectively. The detected B12 in the non-supplemented MacFarlane medium might originate from yeast or meat extract added to the medium. The applied concentrations in our in vitro colon batch (control 5 ng/ml and ExtraB12 2500 ng/ml) are in the range of what we can expect under average diet conditions [average B12 intake of 4.5 μg/day, (16 (link))] or with extreme B12 supplementation [1500 μg per tablet, (10 )]. We therefore considered a small intestine absorption of 50% in the case of average dietary intake (17 ), and 1% in the case of high dose (11 (link), 17 ). Consequently, 2.25 μg of B12 under average diet conditions and 5940 μg of B12 under extreme supplementation conditions will arrive in the proximal colon per day. Taking a proximal colon volume of 200 ml (18 (link)), and a retention time of 8 h (19 (link)), resulting in 600 ml proximal colon suspension per day, which corresponds to the average diet condition of 3.75 ng/ml and with extreme B12 supplementation of 2475 ng/ml.
The flasks were inoculated with the fecal suspension (1% v/v), closed with sterile rubber stoppers and aluminium crimp caps N 20 (Macherey-Nagel AG, Oensingen, Switzerland) and incubated anaerobically at 37°C on a shaker (100 rpm) directly in the anaerobic chamber. After 24 h incubation, 1 ml samples were transferred to a sterile Eppendorf tube and centrifuged at 13,000 × g for 10 min at 4°C. The supernatant was separated from the pellet and filtered through a 0.45 μm nylon membrane filter into a sterile Eppendorf tube. Pellets and supernatants were stored at –20°C until further analysis. For each tested condition and fecal microbiota, fermentations were conducted in triplicates.
Each fecal microbial suspension was grown in three different media containing distinct levels of cyanocobalamin: (i) Control: MacFarlane medium containing normal vitamin solution (5 ng/ml cyanocobalamin); (ii) NB12: no cyanocobalamin added in the vitamin solution used for the MacFarlane medium and (iii) ExtraB12: MacFarlane medium supplemented with high dose of 2500 ng/ml cyanocobalamin final concentration. No B12 was detected in the MacFarlane medium without B12 and in Control medium by UHPLC-DAD method (measured in triplicate; detection limit 30 ng/ml). Therefore, a competitive enzyme immunoassay (RIDASCREEN FAST Vitamin B12, R-Biopharm, Switzerland) was used to quantify B12 content in MacFarlane medium, with and without vitamin mix, quantified as 8.9 ± 0.1 ng/ml and 4.1 ± 0.4 ng/ml, respectively. The detected B12 in the non-supplemented MacFarlane medium might originate from yeast or meat extract added to the medium. The applied concentrations in our in vitro colon batch (control 5 ng/ml and ExtraB12 2500 ng/ml) are in the range of what we can expect under average diet conditions [average B12 intake of 4.5 μg/day, (16 (link))] or with extreme B12 supplementation [1500 μg per tablet, (10 )]. We therefore considered a small intestine absorption of 50% in the case of average dietary intake (17 ), and 1% in the case of high dose (11 (link), 17 ). Consequently, 2.25 μg of B12 under average diet conditions and 5940 μg of B12 under extreme supplementation conditions will arrive in the proximal colon per day. Taking a proximal colon volume of 200 ml (18 (link)), and a retention time of 8 h (19 (link)), resulting in 600 ml proximal colon suspension per day, which corresponds to the average diet condition of 3.75 ng/ml and with extreme B12 supplementation of 2475 ng/ml.
The flasks were inoculated with the fecal suspension (1% v/v), closed with sterile rubber stoppers and aluminium crimp caps N 20 (Macherey-Nagel AG, Oensingen, Switzerland) and incubated anaerobically at 37°C on a shaker (100 rpm) directly in the anaerobic chamber. After 24 h incubation, 1 ml samples were transferred to a sterile Eppendorf tube and centrifuged at 13,000 × g for 10 min at 4°C. The supernatant was separated from the pellet and filtered through a 0.45 μm nylon membrane filter into a sterile Eppendorf tube. Pellets and supernatants were stored at –20°C until further analysis. For each tested condition and fecal microbiota, fermentations were conducted in triplicates.
Corresponding organizations : ETH Zurich
5
Yeast Extract-Based Media Preparation
Yeast extract was purchased from Bioferm (Waldmünchen, Germany). Bactotryptone was supplied by Difco (Detroit, Mich.). Casein peptone and protease peptone were obtained from Merck (Darmstadt, Germany), as was D- glucose. Most amino acids were from Ajinomoto (Hamburg, Germany), only asparagine was obtained from ICN (MP Biomedicals, Eschwege, Germany), histidine from Senn Chemicals (Dielsdorf, Switzerland), and glycine from Carl Roth (Karlsruhe, Germany). Folic acid and dihydrostreptomycin sulfate were supplied by Sigma (St. Louis, Mo.), whereas geneticin (G-418) was from Serva (Heidelberg, Germany). Cyanocobalamin, lipoic acid, riboflavin, thiamine-HCl, biotin were purchased from Merck. All other chemicals were at least of analytical grade.
Corresponding organizations : Xiamen University, University of Groningen, Bielefeld University
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