Acetylcholine chloride, (-)-nicotine tartrate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), amphotericin B, penicillin/streptomycin, protease type XIV, collagenase Type I, poly-D-lysine hydrobromide, Red Blood Cell Lysis solution, dimethylsulfoxide, and all salts were purchase from Sigma-Aldrich (St. Louis, MO, USA). Varenicline tartrate and tetrodotoxin citrate (TTX) were purchased from Tocris Bioscience (Abindgon, UK). Dulbecco's Modified Eagle's Medium (DMEM) and Glutamax were purchased from Life Technologies (Carlsbad, CA, USA). Fetal bovine serum was from LabClinics (Barcelona, Spain) and the D-glucose from Panreac (Barcelona, Spain).
D glucose
Manufactured by ITW Reagents
Sourced in Spain
D-glucose is a monosaccharide, a type of simple sugar that serves as a primary source of energy for many organisms. It is a clear, colorless, crystalline solid that is soluble in water and various organic solvents. D-glucose plays a crucial role in various biochemical processes and is an essential component of many laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by ITW Reagents (6 mentions)
Methanol, by ITW Reagents (6 mentions)
Potassium hexacyanoferrate 2 trihydrate, by Merck Group (5 mentions)
13c3 labelled acrylamide, by Cambridge Isotopes (5 mentions)
Zinc acetate dehydrate, by Merck Group (4 mentions)
Yeast extract, by Helicon (3 mentions)
Carrez, by Merck Group (3 mentions)
Milli q integral 5 water purification system, by Merck Group (3 mentions)
D fructose, by ITW Reagents (3 mentions)
Na2hpo4, by Dia-M (2 mentions)
Ferrocene, by Dia-M (2 mentions)
Sucrose, by ITW Reagents (2 mentions)
Hexane, by ITW Reagents (2 mentions)
Ethanol, by ITW Reagents (2 mentions)
D sorbitol, by ITW Reagents (2 mentions)
Integral 5 water purification system, by Merck Group (2 mentions)
D galactose, by Merck Group (2 mentions)
Acetylcholine chloride, by Merck Group (1 mentions)
Red blood cell lysis solution, by Merck Group (1 mentions)
Nicotine tartrate, by Merck Group (1 mentions)
19 protocols using d glucose
1
Primary Cell Culture Isolation
2
Carbohydrate Analysis of Cell Walls
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The cellulose content of CWs was quantified by the Updegraff method [103 (link)], under the hydrolytic conditions described by Saeman, [104 ], using glucose as standard.
The total sugars and uronic acids were determined by the phenol-sulfuric acid method [105 (link)], and the m-hydroxydiphenyl method [106 (link)], using D-glucose and galacturonic acid as reference, respectively. For the neutral sugar estimation, the values for total sugars and uronic acids were subtracted.
For the neutral sugar composition, samples from each fraction were hydrolyzed with 2 N TFA for 1 h at 121 °C, which resulted in monosaccharides that were derivatized to alditol acetates following the method described by Albersheim [107 (link)]. Furthermore, the alditol acetates were quantified by gas chromatography (GC) using a Perkin-Elmer equipment with a flame ionization detector (GC-FID), using a Supelco SP-2330 column and a Perkin-Elmer GC-FID, as described in Rebaque et al. (2017) [61 (link)]. Inositol was used as internal control, and monosaccharides L(−)rhamnose (Merck), L(−)fucose (Sigma), L(+)arabinose (Merck), D(+)xylose (Merck), D(+)mannose (Merck), D(+)galactose (Merck), and D(+)glucose (Panreac) as standard markers.
The total sugars and uronic acids were determined by the phenol-sulfuric acid method [105 (link)], and the m-hydroxydiphenyl method [106 (link)], using D-glucose and galacturonic acid as reference, respectively. For the neutral sugar estimation, the values for total sugars and uronic acids were subtracted.
For the neutral sugar composition, samples from each fraction were hydrolyzed with 2 N TFA for 1 h at 121 °C, which resulted in monosaccharides that were derivatized to alditol acetates following the method described by Albersheim [107 (link)]. Furthermore, the alditol acetates were quantified by gas chromatography (GC) using a Perkin-Elmer equipment with a flame ionization detector (GC-FID), using a Supelco SP-2330 column and a Perkin-Elmer GC-FID, as described in Rebaque et al. (2017) [61 (link)]. Inositol was used as internal control, and monosaccharides L(−)rhamnose (Merck), L(−)fucose (Sigma), L(+)arabinose (Merck), D(+)xylose (Merck), D(+)mannose (Merck), D(+)galactose (Merck), and D(+)glucose (Panreac) as standard markers.
3
Microbial Biosensor Characterization
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The nutrient medium was prepared using D-glucose (Panreac, Barcelona, Spain), peptone (Condra, Spain), tryptone (Condra, Spain), and yeast extract (Helicon, Moscow, Russia).
Biosensor measurements were performed using a sodium-potassium phosphate buffer solution pH = 6.8 (33 mM KH2PO4 + 33 mM Na2HPO4, Dia-m, Moscow, Russia).
All other reagents and solvents used were chemically pure and produced by ChemMed (Moscow, Russia) and Sigma–Aldrich (Moscow, Russia).
Biosensor measurements were performed using a sodium-potassium phosphate buffer solution pH = 6.8 (33 mM KH2PO4 + 33 mM Na2HPO4, Dia-m, Moscow, Russia).
All other reagents and solvents used were chemically pure and produced by ChemMed (Moscow, Russia) and Sigma–Aldrich (Moscow, Russia).
4
Candida Species Inoculation in G. mellonella
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Six commercially available reference strains, including two strains of each species C. glabrata, C. nivariensis and C. bracarensis, were obtained from different culture collections (Table 1 ).
Yeasts were cultured overnight in yeast extract peptone dextrose broth (YEPD; 1% yeast extract, 2% bacteriological peptone, 2% d-glucose) medium (Panreac, Spain) at 30 °C under shaking conditions. Then, yeast cells were washed three times with phosphate-buffered saline solution (PBS) and resuspended in PBS supplemented with ampicillin (20 mg/L) to prevent infection with bacteria naturally present on the surface of G. mellonella larvae. Cell counting was performed by microscopy using a Burker haemocytometer and three concentrations of 1 × 107, 1 × 108 and 1 × 109 yeast cells/mL were prepared in PBS-ampicillin (20 mg/L) to use as inocula.
Yeasts were cultured overnight in yeast extract peptone dextrose broth (YEPD; 1% yeast extract, 2% bacteriological peptone, 2% d-glucose) medium (Panreac, Spain) at 30 °C under shaking conditions. Then, yeast cells were washed three times with phosphate-buffered saline solution (PBS) and resuspended in PBS supplemented with ampicillin (20 mg/L) to prevent infection with bacteria naturally present on the surface of G. mellonella larvae. Cell counting was performed by microscopy using a Burker haemocytometer and three concentrations of 1 × 107, 1 × 108 and 1 × 109 yeast cells/mL were prepared in PBS-ampicillin (20 mg/L) to use as inocula.
5
Biofilm Cultivation Media and Graphite Paste Electrode
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D-glucose (Panreac, Barcelona, Spain), peptone (Condra, Barcelona, Spain), tryptone (Condra, Barcelona, Spain), and yeast extract (Helicon, Moscow, Russia) were applied as nutrients for biofilm cultivation. Graphite powder (Fluka, Berlin, Germany), paraffin oil (Fluka, Berlin, Germany), ferrocene (Dia-m, Moscow, Russia), and a dialysis membrane with a transmission limit of 14 kDa (Roth, Dautphetal, Germany) were used for working graphite paste electrode (GPE) formation. Sodium–potassium phosphate buffer solution, pH = 6.8, was prepared from 33 mM KH2PO4 and 33 mM Na2HPO4 (Dia-m, Moscow, Russia).
6
Quantitative Acrylamide Isotope Analysis
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Potassium hexacyanoferrate(II) trihydrate (98%, Carrez-I) and zinc acetate dihydrate (>99%, Carrez-II) were obtained from Sigma (St Louis, USA). 13 (link) C 3 -Labelled acrylamide (99% isotopic purity) was obtained from Cambridge Isotope Laboratories (Andover, MA, USA). Formic acid (98%), D(+)-glucose and methanol (99.5%) were from Panreac (Barcelona, Spain). Deionized water was obtained from a Milli-Q Integral 5 water purification system (Millipore, Billerica, MA, USA). All other chemicals, solvents and reagents were of analytical grade.
7
Spectrophotometric Enzyme Activity Assay
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Reagents: Sodium dihydrogen phosphate (99.99%) and di-sodium hydrogen phosphate anhydrous (99.99%) were acquired from Merck (Steinheim, Germany). o-dianisidine dihydrochloride (D9154), horseradish peroxidase (P8375-5KU), hydrogen peroxide (>30%), and dialysis membrane (D6191) were acquired from Sigma-Aldrich (St. Louis, MO, USA), which is a part of Merck. Potassium permanganate, sodium oxalate, hydrochloric acid, Baird Parker agar base (BP), and egg yolk emulsion with potassium tellurite were acquired from VWR International Eurolab, which is part of Avantor (Llinars del Vallés, Cataluña, Spain). D-(+)-Glucose (99.99%) was acquired from Panreac (Barcelona, Spain). Nutrient broth no. 2 (NB) and Ringer’s solution were acquired from Oxoid, which is part of Thermo Fisher (Basingstoke, Hampshire, UK). Water was deionized using a Milli-Q water purification system (Wasserlab, Navarra, Spain).
Apparatus: We used the Varian Cary Bio 400 spectrophotometer (Varian, part of Agilent Technologies, Santa Clara, CA, USA) and fluorometer Varioskan LUX microplate reader (Thermo Fisher, Kanderl, Germany). We also used sterile 96-well round-bottomed polystyrene microtiter plates (Brand, Wertheim, Germany).
Apparatus: We used the Varian Cary Bio 400 spectrophotometer (Varian, part of Agilent Technologies, Santa Clara, CA, USA) and fluorometer Varioskan LUX microplate reader (Thermo Fisher, Kanderl, Germany). We also used sterile 96-well round-bottomed polystyrene microtiter plates (Brand, Wertheim, Germany).
8
Freeze-Drying Optimization of Lactic Acid Bacteria
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The LAB cultures left in MRS broth overnight were centrifuged at 4000 x g for 10 min at 4 °C. The pellets were washed twice using saline solution (0.9% NaCl) and suspended in 2 ml of cryoprotectant solutions. Three cryoprotectants (at a final concentration of 2%) were tested: D-sorbitol (Sigma-Aldrich, Saint Louis, MO, USA), D-glucose, and sucrose (PanReac AppliChem, Darmstadt, Germany). After freezing at -20 °C overnight, cell suspensions (prepared as described above) were freeze-dried in a chamber-type freeze-dryer (FreeZone6, LABCONCO, 6 L Benchtop Freeze Dry System, Kansas, MO, USA) at − 55 °C and 0.3 mbar, for 4 h. The cell viability was tested before and after the freeze-drying procedure by the plate count method. Distilled water was used as a control. The survival rate of LAB strains was calculated as:
, where N0 and N mean the viable cells (CFU/ml) before and after freeze drying, respectively.
, where N0 and N mean the viable cells (CFU/ml) before and after freeze drying, respectively.
9
Ferrocene-mediated Biosensor Development
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Ferrocene (Dia-m, Moscow, Russia) was used as an electron transport mediator. Nutrient medium was prepared using D-glucose (Panreac, Barcelona, Spain), peptone (Conda, Madrid, Spain), tryptone (Conda, Madrid, Spain), and yeast extract (Helicon, Moscow, Russia). Graphite powder with a particle size of 75 μm with a high frequency of 99.997% (Fluka, Darmstadt, Germany), paraffin oil (Fluka, Germany), carbon nanotubes of the “Taunit” («NanoTechCenter», Tambov, Russia) series with an outer diameter of 10–30 nm, length ≥ 2 μm, and amount of impurities ≤ 1%, and a dialysis membrane with a transmission limit of 14 kDa (Roth, Germany) were taken to create a working graphite paste electrode. Biosensor measurements were performed using a sodium-potassium phosphate buffer solution pH = 6.8 (33 mM KH2PO4 + 33 mM Na2HPO4, Dia-m, Moscow, Russia).
10
Quantitative Analysis of Carbohydrates
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Potassium hexacyanoferrate (II) trihydrate (98%, Carrez-I) and zinc acetate dehydrate (>99%, Carrez-II) were obtained from Sigma (St. Louis, MO, USA). 13C3-labelled acrylamide (99% isotopic purity) was obtained from Cambridge Isotope Laboratories (Andover, MA, USA. Formic acid (98%), D(+) glucose, D(-) fructose, D(-) sorbitol, ethanol, methanol (99.5%) and hexane were obtained from Panreac (Barcelona, Spain). Deionized water was obtained from a Milli-Q Integral 5 water purification system (Millipore, Billerica, MA, USA). All other chemicals, solvents and reagents were of analytical grade.
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