Calu-3, a human submucosal gland cell line derived from bronchial adenocarcinoma, was purchased from Elabscience Biotechnology Inc. (USA). Calu-3 cells were cultured at 37 °C with 5% CO2 in MEM (Nacalai Tesque, Inc., Kyoto, Japan) with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque, Inc.). The cells were stimulated with different D-glucose concentrations as follows: 100 mg/dL D-glucose (Nacalai Tesque, Inc.) in MEM (NG) and 1000 mg/dL D-glucose in MEM (HG). The medium was changed every alternate day. BAY-876 (Medchemexpress, Monmouth Junction, NJ, USA) was used to inhibit GLUT1.
D glucose
Manufactured by Nacalai Tesque
Sourced in Japan
D-glucose is a type of monosaccharide, which is the simplest form of carbohydrate. It serves as a primary source of energy for many organisms and is a key component in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Nahco3, by Nacalai Tesque (2 mentions)
Bacto peptone, by BD (2 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Minimum essential medium (mem), by Nacalai Tesque (1 mentions)
Bay 876, by MedChemExpress (1 mentions)
Yeast extract, by BD (1 mentions)
Hemin, by Merck Group (1 mentions)
L cysteine hydrochloride monohydrate, by Merck Group (1 mentions)
Anaeropack system, by Mitsubishi (1 mentions)
Hydroxychloroquine sulfate hcq, by Merck Group (1 mentions)
D mannose, by Nacalai Tesque (1 mentions)
Milli q purification system, by Merck Group (1 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
Oleic acid, by Nacalai Tesque (1 mentions)
Rhodamine b 1 2 dihexadecanoyl sn glycero 3 phosphoethanolamine, by Thermo Fisher Scientific (1 mentions)
Prism 9, by GraphPad (1 mentions)
Dmem high glucose, by Fujifilm (1 mentions)
Hyperflask, by Corning (1 mentions)
18 protocols using d glucose
1
Calu-3 Cell Culture and Glucose Stimulation
2
Anaerobic Culturing of Oral Bacteria
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The bacterial strains used in this study were Fusobacterium nucleatum ATCC25586 (FN), Actinomyces naeslundii X600 (AN) and Streptococcus mitis ATCC 903 (SM). These bacterial strains were cultured anaerobically in BHIS broth using the AnaeroPack System (Mitsubishi Gas Chemical Company, Inc.). BHIS broth is Brain Heart Infusion medium (Eiken Chemical Co.) supplemented with 5 µg/ml hemin (Sigma-Aldrich), 0.1% L-cysteine hydrochloride monohydrate (Sigma-Aldrich), 0.5% yeast extract (Becton Dickinson and Company) and 0.375% D-glucose (Nacalai Tesque).
3
Monosaccharide Characterization for Research
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The monosaccharides used in this study are listed in Table I . D-glucose was purchased from Nacalai Tesque, and D-mannose, D-allose, D-psicose, D-tagatose, D-sorbose, L-psicose, D-arabinose, L-arabinose, and L-fucose were obtained from Matsutani Chemical Industry Co., Ltd. Hydroxychloroquine (HCQ) sulfate was purchased from Sigma-Aldrich/Merck KGaA (catalog no. H0915).
4
Phospholipid and Fatty Acid Analysis
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The saturated phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the unsaturated phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (Chol) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammonium salt; Rho-DHPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitrobenz-2-1,3-benzoxadiazol-4-yl) (ammonium salt; NBD-PE) were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and Avanti Polar Lipids, respectively. Oleic acid (C18:1, cis-9; OA), palmitOleic acid (C16:1, cis-9; PaA), and d -(+)-glucose were acquired from Nacalai Tesque (Kyoto, Japan). Eicosenoic acid (C20:1, cis-9; EiA) was obtained from Larodan Fine Chemicals (Malmö, Sweden). Ultrapure water (specific resistivity ≥ 18 MΩ·cm) was obtained from a Millipore Milli-Q purification system (Burlington, MA, USA). The chemical structures of the lipids and MUFAs used in this study are shown in Figure S1 .
5
Oral Glucose Tolerance Test for Diabetes
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The OGTT was conducted weekly from the time of STZ administration until the mice were euthanized to confirm complete induction of T2DM. All of the mice were fasted overnight for 18 h prior to the test56 (link). Baseline blood glucose levels were measured at 0 min, and a 10% glucose solution prepared with d -(+)-glucose (Nacalai Tesque, Japan) in distilled water was orally administered at a dose of 1 g/kg body weight57 (link) and a volume of 10 μl/g body weight58 (link). Blood glucose levels were recorded at 0, 30, 60, 90 and 120 min59 (link) using the ACCU-CHEK Guide Blood Sugar Test Kit (Roche Diagnostics, Indianapolis, IN, USA). Blood samples (5 μl) were obtained by tail pricking. The area under the curve for blood glucose (AUCglu) were calculated using GraphPad Prism 9 software (GraphPad Software Inc., La Jolla, CA, USA).
6
Large-scale AAV vector production
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AAV vectors were prepared in a large scale and harvested from culture supernatant (conditioned media), as previously described [3 (link)]. In brief, a 293EB cell line expressing adenoviral E1a, adenoviral E1b, and Bcl-xL [15 (link)] was expanded in two 500 mL flasks (HYPERFlask, Corning, Corning, NY, USA) or a 1 L bioreactor (iCELLis Nano Bioreactor, Pall, Port Washington, NY, USA) for 5 days or 4 days, respectively, in Dulbecco’s Modified Eagle Medium (DMEM high glucose, FUJIFILM Wako, Chuo-ku, Osaka, Japan) with 10% fetal bovine serum (Thermo Fisher, Waltham, MA, USA). Transfection was then performed with polyethylenimine max, (Polysciences, Warrington, PA, USA) using pAAV-ZsGreen1 (TaKaRa Bio, Kusatsu, Shiga, Japan), pRC9 (serotype 9), and helper plasmids in DMEM including 2 mM L-Alanyl-L-glutamine Solution(100x) (Nacalai Tesque, Nakagyo-ku, Kyoto, Japan), 0.12% NaHCO3 (Nacalai Tesque), and 0.13% D-glucose (Nacalai Tesque) without serum. Five days post-transfection, culture supernatants were harvested and treated with 18.5 U/mL endonuclease (KANEKA CORPORATION, Minato-ku, Tokyo, Japan) with 5 mM MgCl2 (Nacalai Tesque) for 30 min at 37 °C. All cells were checked for mycoplasma contaminations resulting were reported negative.
7
Transglucosylation Kinetics of Mutant Sucrose Enzymes
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A reaction mixture (1 mL) containing enzymes (9.8 μM wild type, 0.8 μM N258P, or 12 μM N258L), 100 mM sucrose, and 10 mM MES-NaOH buffer (pH 6.0) was incubated at 37 °C. Aliquots (0.14 mL) were taken from the reaction mixture at indicated times and heated at 100 °C for 5 min. Five micrograms of carbohydrate was spotted on TLC plate silica gel 60 F254 (Merck, Darmstadt, Germany). A developing solvent, chloroform/acetic acid/water (6/7/1, v/v/v), was used and the carbohydrates were visualized by spraying with the detection reagent, 85% orthophosphoric acid/0.01% aniline in acetone (10/1, v/v), and heating the TLC plate.
The transglucosylation product from sucrose by N258P was analyzed by HPAEC-PAD. A reaction mixture (0.5 mL) containing 0.49 μM N258P, 100 mM sucrose, and 10 mM MES-NaOH buffer (pH 6.0) was incubated at 37 °C for 30 min. The reaction was stopped by heating at 100 °C for 5 min. HPAEC-PAD analysis was performed as described above, but 480 mM NaOH was used as the eluant.d -Glucose, d -fructose (Nacalai Tesque), sucrose, and erlose (Seikagaku, Tokyo, Japan) (200 μM) were used as standards.
The transglucosylation product from sucrose by N258P was analyzed by HPAEC-PAD. A reaction mixture (0.5 mL) containing 0.49 μM N258P, 100 mM sucrose, and 10 mM MES-NaOH buffer (pH 6.0) was incubated at 37 °C for 30 min. The reaction was stopped by heating at 100 °C for 5 min. HPAEC-PAD analysis was performed as described above, but 480 mM NaOH was used as the eluant.
8
Glucose Tolerance Testing in Mice
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IPGTT was performed as described previously90 (link) with modification. Briefly, mice were fasted overnight (16 h) and then intraperitoneally injected with d -(+)-glucose (2 g/kg body weight; 20% solution, Nacalai Tesque, Kyoto, Japan). To measure the blood glucose level, blood was obtained from the tail vein by cutting with a single-edged blade (Feather, Osaka, Japan) and analyzed on a ONE TOUCH Ultra Vue (LifeScan Japan, Tokyo, Japan) before and after glucose injection at the indicated time points. Blood insulin levels were measured by using an LBIS Mouse Insulin ELISA kit (Wako Pure Chemicals, Osaka, Japan) in accordance with the manufacturer’s instructions. Samples unsuitable for analysis, such as hemolyzed blood, were excluded.
9
Antimicrobial and Wound Healing Potential of Tiger Milk Mushroom and Pomegranate
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L. rhinocerotis (Tiger Milk Mushroom) sclerotia powder was obtained as a gift from Lignas Bio Synergy Plt., Selangor, Malaysia). P. granatum fruits were purchased from a supplier in Kuala Lumpur, Malaysia. Pluronic F-127 was purchased from Sigma-Aldrich (Missouri, USA). PEG 400 with an average molecular mass of 380–420 g/mol was procured from Merck (Darmstadt, Germany). For the antibacterial test, Staphylococcus aureus (ATCC 6538) was obtained from the Microbiology Laboratory of Faculty Pharmacy, Universiti Kebangsaan Malaysia, Kuala Lumpur. Mueller–Hinton broth (MHB) and Mueller–Hinton agar (MHA) were purchased from the Difco laboratory of Becton Dickinson Company, USA. Gentamicin sulphate was purchased from Nascalai Tesque, Kyoto, Japan. SensoLyte® 520 Thrombin Assay Kit *Fluorometric* was purchased from AnaSpec (California, USA). Human dermal fibroblasts (HDFs, passage 2–8) were used for cell migration assays. Dulbecco’s modified Eagle’s medium (DMEM) (high-glucose) comprising 4.5 g/L of D-(+)-Glucose, L-Glutamine, phenol red, HEPES, and sodium pyruvate was obtained from Nacalai Tesque (Kyoto, Japan). A solution of 0.05% Trypsin/0.53 mM EDTA was also procured from Nacalai Tesque (Kyoto, Japan). Pen-Strep (penicillin streptomycin) and fetal bovine serum (FBS) were purchased from Gibco (USA), while phosphate-buffered saline (PBS) was obtained from Invitrogen (USA).
10
Bovine Oocyte In Vitro Fertilization
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After in vitro maturation, the COCs were transferred into a 15 ml tube containing 2 ml of HEPES-TLP-PVA supplemented with 0.1% (w/v) hyaluronidase (Sigma) and vortexed for
90 sec to remove cumulus cells from oocytes. Denuded oocytes were transferred to droplets (80 µl) of fertilization medium in groups of 15 to 20 under paraffin oil in a 35 mm polystyrene
dish. The fertilization medium was composed of 114.0 mM NaCl (Nacalai), 3.2 mM KCl (Nacalai), 6.76 mM CaCl2·2H2O (Nacalai), 0.5 mM MgCl2·6H2O
(Nacalai), 0.1 mM sodium pyruvate, 10.0 mM sodium lactate (Sigma), 0.35 mM NaH2PO4·2H2O (Nacalai), 5.0 mM D-glucose, 25.0 mM NaHCO3 (Nacalai),
0.3% (w/v) bovine serum albumin (Fraction V; Sigma), 100 µg/ml amikacin sulfate, and 2.0 mM caffeine (Sigma). The frozen spermatozoa were thawed immediately before insemination, as described
above. HEPES-TLP-PVA containing frozen-thawed spermatozoa was centrifuged at 760 ×g for 10 min at 38°C, and the supernatant was removed. The precipitated spermatozoa were
gently suspended in the fertilization medium at a concentration of 3.5 × 107 spermatozoa/ml, and 20 µl of this sperm suspension was introduced into the 80 µl droplet that
contained denuded oocytes at a final concentration of 7.0 × 106 spermatozoa/ml. The oocytes and spermatozoa were then cultured for 12 h at 38.5°C in an atmosphere with 5%
CO2 in air.
90 sec to remove cumulus cells from oocytes. Denuded oocytes were transferred to droplets (80 µl) of fertilization medium in groups of 15 to 20 under paraffin oil in a 35 mm polystyrene
dish. The fertilization medium was composed of 114.0 mM NaCl (Nacalai), 3.2 mM KCl (Nacalai), 6.76 mM CaCl2·2H2O (Nacalai), 0.5 mM MgCl2·6H2O
(Nacalai), 0.1 mM sodium pyruvate, 10.0 mM sodium lactate (Sigma), 0.35 mM NaH2PO4·2H2O (Nacalai), 5.0 mM D-glucose, 25.0 mM NaHCO3 (Nacalai),
0.3% (w/v) bovine serum albumin (Fraction V; Sigma), 100 µg/ml amikacin sulfate, and 2.0 mM caffeine (Sigma). The frozen spermatozoa were thawed immediately before insemination, as described
above. HEPES-TLP-PVA containing frozen-thawed spermatozoa was centrifuged at 760 ×g for 10 min at 38°C, and the supernatant was removed. The precipitated spermatozoa were
gently suspended in the fertilization medium at a concentration of 3.5 × 107 spermatozoa/ml, and 20 µl of this sperm suspension was introduced into the 80 µl droplet that
contained denuded oocytes at a final concentration of 7.0 × 106 spermatozoa/ml. The oocytes and spermatozoa were then cultured for 12 h at 38.5°C in an atmosphere with 5%
CO2 in air.
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