Flp-In-HEK293 cells stably transformed with the methylated or unmethylated EDI minigene were cultured in complete DMEM medium with high glucose (Sigma), 10% fetal bovine serum (Sigma), 2 mg/mL l -alanyl-l -glutamine (Biological Industries Israel), 100 U/mL penicillin, 0.1 mg/mL streptomycin (Biological Industries Israel) supplemented with 100 µg/mL hygromycin B (InvivoGen). HCT 116 human colon carcinoma cells were cultured in complete RPMI medium with high glucose (Sigma), 10% fetal bovine serum (Sigma), 2 mg/mL l -alanyl-l -glutamine (Biological Industries Israel), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Biological Industries Israel). All cells were grown at 37°C in a humidified atmosphere with 5% CO2.
High glucose
Manufactured by Merck Group
Sourced in United States, Germany, Israel
High glucose is a laboratory reagent used to measure glucose levels in various biological samples. It serves as a standard solution for the calibration and verification of glucose measurement devices and assays.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Merck Group (4 mentions)
Penicillin, by Merck Group (4 mentions)
L glutamine, by Merck Group (4 mentions)
Glutamine, by Merck Group (3 mentions)
3 iaid, by Merck Group (3 mentions)
D glucose, by Merck Group (3 mentions)
Low glucose, by Merck Group (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Amphotericin b as fungizone, by Merck Group (2 mentions)
Penicillin, by Sartorius (1 mentions)
Streptomycin, by Sartorius (1 mentions)
L alanyl l glutamine, by Sartorius (1 mentions)
Hygromycin b, by InvivoGen (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Halo tag r110 direct ligand, by Promega (1 mentions)
44 protocols using high glucose
1
Methylation Effects on EDI Minigene Expression
2
Visualizing Histone and MeCP2 in C2C12 Cells
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C2C12 mouse myoblast cells (see Table S3 ) were grown in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (#D6429, Sigma-Aldrich) supplemented with 20% fetal bovine serum (#F7524, Sigma-Aldrich), 1× glutamine (#G7513, Sigma-Aldrich), and 1 μM gentamicin (#G1397, Sigma-Aldrich) at 37 °C and 5% CO2 in a humidified incubator. Tests to check for potential mycoplasma contamination were performed regularly.
The C2C12 cells were cotransfected with plasmids expressing histone GFP-H1 [57 (link)] and MeCP2-Halo (seeTable S5 ) at different ratios (2:1, 1:1, 1:2) using the Neon transfection System (Thermo Fisher Scientific) according to the manufacturer’s instructions. Voltage, width, and pulse for C2C12 cells were: 1650 V, 10 ms, and 3 pulses. Then, cells were seeded onto coverslips and grown at 37 °C and 5% CO2 in a humidified incubator. Twenty-four hours after transfection, Halo Tag R110 Direct Ligand (G 3221, Promega GmbH, Walldorf, Germany) was added to the medium to visualize MeCP2.
The C2C12 cells were cotransfected with plasmids expressing histone GFP-H1 [57 (link)] and MeCP2-Halo (see
3
Quantifying Cellular Oxidative Stress
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The production of ROS was evaluated by analyzing the fluorescence intensity by dihydroethidium (DHE; Invitrogen), DCFDA (Abcam), and MitoSOX (Invitrogen) staining. In brief, frozen musculus gastrocnemius sections were stained with 5 μM DHE at 37°C for 30 min, and then fluorescence intensity was detected by confocal microscopy. HUVECs were stimulated with high glucose (25 mM; Sigma–Aldrich; 20 (link)) or 3-IAId (0.5 mM; Sigma–Aldrich; 5 (link)) for 24 h, subjected to DCFDA (20 μM), or MitoSOX (5 μM) staining at 37°C for 30 min and then assessed by fluorescence microscopy.
4
Mammalian Cell Culture and Transfection Protocols
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HEK293, COS-7 and U-2OS cells were cultured in Dubecco’s modified Eagle’s medium (DMEM), high glucose (Sigma-Aldrich) supplemented with 3.7 g/l sodium bicarbonate and 10% FBS. Caco-2 and TC-7 cells were grown in DMEM, high glucose supplemented with 3.7 g/l sodium bicarbonate, 1% non-essential amino acids (NEAA) and 20% FBS. A2780, HL-60 and HL-60/MX2 cells were maintained in medium RPMI 1640 (Sigma-Aldrich) supplemented with 2 g/l sodium bicarbonate and 10% FBS. A2780cisR cells was cultured in RPMI 1640 medium supplemented with 2 g/l sodium bicarbonate, 10% FBS and 1 μM cisplatin. All mammalian cells were grown in a humidified incubator with 5% CO2 at 37 °C. Transfection was carried out using Lipofectamine 2000 according to the manufacture’s protocol (Thermo Fisher Scientific).
5
HUVEC Cultures for Glucose Metabolism
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Human umbilical cord vein endothelial cells (HUVECs) obtained from Lonza were grown in F-12K medium (7 mM glucose present in medium) supplemented with 30 μg/mL endothelial cell growth supplement (ECGS, Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/mL heparin (Alfa Aesar, Stoughton, MA, USA). Cells were cultured in 75 cm2 flasks, 96-well plates, 24-well plates and 6-well plates precoated with 0.2% gelatin and maintained at 37 °C in a humidified atmosphere of 5% CO2. Medium was changed every two days and passaged by 0.5% trypsin-EDTA (Gibco, Grand Island, NY, USA). HUVECs at 4–8 passages were used for experiments. HUVECs were treated with mannitol as osmotic control, high glucose (30 mM, 48 h; Sigma-Aldrich, St. Louis, MO, USA) and cotreated with JAT (1 µM).
6
Chondrogenic Differentiation of MSCs
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MSCs were trypsinized and seeded in V-bottomed 96-well polypropylene plates at 200,000 cells per well. Pellets were formed by centrifugation at 250 g for 5 min and chondrogenically differentiated in DMEM with high glucose (Invitrogen), 1% ITS (Sigma Aldrich), 50 μg/ml ascorbate-2-phosphate (Sigma Aldrich), 40 μg/ml L-proline (Sigma Aldrich), 100 nM dexamethasone (Sigma Aldrich), 1 mM sodium pyruvat (Invitrogen) and 10 ng/ml TGFβ1 (R&D Systems).
After a pre-differentiation period of 14 days, medium conditions were changed to hypertrophy enhancing medium (hyp) consisting of DMEM high glucose, 1% ITS, 50 g/ml ascorbate-2-phosphate, 40 g/ml L-proline, 1 nM triiodothyronine (T3) (Sigma Aldrich) and the control was kept in chondrogenic medium (chon) for the whole culture period. The medium was changed three times per week.
Aggregates were harvested at d1, d3, d7, d14, d17, d21 and d28 for gene expression analysis. Aggregates for histological analysis were harvested on d14 and d28.
After a pre-differentiation period of 14 days, medium conditions were changed to hypertrophy enhancing medium (hyp) consisting of DMEM high glucose, 1% ITS, 50 g/ml ascorbate-2-phosphate, 40 g/ml L-proline, 1 nM triiodothyronine (T3) (Sigma Aldrich) and the control was kept in chondrogenic medium (chon) for the whole culture period. The medium was changed three times per week.
Aggregates were harvested at d1, d3, d7, d14, d17, d21 and d28 for gene expression analysis. Aggregates for histological analysis were harvested on d14 and d28.
7
Caco-2 Cell Culture and Supernatant Preparation
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The Caco-2 cells were routinely grown at 37°C in a 95% air–5% CO2 atmosphere in Dulbecco's modified Eagle medium (DMEM) with high glucose (Sigma-Aldrich Co) and 10% heat-inactivated (56°C, 30 min) fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The cells were harvested using trypsin-EDTA (Gibco) and suspended with DMEM-10% FBS to stop the reaction with trypsin-EDTA. The cells were collected from the cell suspension by a centrifugation, and were washed with Dulbecco's phosphate buffered saline (DPBS; Gibco). Then, the cells were resuspended with DMEM without any serum, and the mixture was centrifuged twice. Finally, the cellular suspension in DMEM was adjusted to 1.0 × 105 cells per 75 cm2 of the tissue culture flask (Thermo Scientific, Waltham, MA, USA). The culture medium was replaced every 5 days, and the supernatant collected after 5, 10, and 15 days of culture was used in subsequent experiments. The collected supernatant was filtered with Centriprep YM-3 or YM-10 (3 or 10 kDa; Millipore, Bedford, MA, USA). For some experiments, 500 μl of the various Caco-2 cell supernatant samples were treated with 25 μl of Immobilized TPCK Trypsin (Thermo Fisher) for 1 h at 37°C; the Immobilized TPCK Trypsin was then removed by centrifugation (10,000 × g for 10 min).
8
Culturing Human Keratinocyte Cells
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As material in our work, human keratinocyte cells (HaCaT; CLS Cell Lines Service, Eppelheim, Germany) were used. The cell culture was carried out with the use of Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with high glucose (4500 mg/L, Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), penicillin (100 U/mL; Sigma-Aldrich, St. Louis, MO, USA), streptomycin (100 mg/mL; Sigma-Aldrich, St. Louis, MO, USA), and glutamine (2 mm; Sigma-Aldrich, St. Louis, MO, USA). The keratinocytes were incubated with a 5% CO2 enriched atmosphere and at a constant temperature of 37 °C.
9
Isolation and Purification of H- and L-Particles from HSV-1-Infected Cells
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H- and L-particles were isolated from supernatants derived from HSV-1-infected BHK21 cells as described in “Amplification of virus strains.” The isolation of H- and L-particles was performed according to a previously published protocol (47 (link)). Briefly, a gradient of 5 to 20% Ficoll PM 400 (Sigma-Aldrich, Germany) was loaded with the virus suspension and centrifuged at 26,000 × g for 2 h at 4°C. The H- and L-particle bands were harvested via punctuation with a needle, transferred into centrifugation tubes (Beckman Coulter, USA) and were filled up with 30 mL DMEM without phenol red (high glucose, Sigma-Aldrich, Germany). Both particle types were centrifuged at 80,000 × g for 2 h at 4°C. For further usage, particles were resuspended in an appropriate amount of DMEM without phenol red depending on the pellet size and stored at −80°C. In order to inactivate contaminating H-particles, the L-particle preparations were UV-irradiated three times applying 0.12 J/cm2 in a Vilber Luormat (Biometra, Germany).
10
Apelin-55 Stimulation and Inhibition
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Following the final step of cell setup (HEK293A transfection or 3T3-L1 differentiation), His-apelin-55 was dissolved (3 μg/μL; 358 μM) in phenol red-free DMEM (High Glucose, Sigma-Aldrich). Medium was replaced with serum-free and phenol red-free DMEM (High Glucose, 400 μL/well). Each well of the plate was supplemented with dissolved protein (50 μL) at a final concentration of 0.333 μg/μL per well. Cells were exposed to His-apelin-55 for designated time points. For inhibition studies, cells were exposed to decanoyl-RVKR-CMK (Biomol International) in serum-free and phenol red-free DMEM (High Glucose, 400 μL/well) at concentrations of 25, 2.5, or 0.25 μM for 1 h prior to supplementation with His-apelin-55. For 0 h (control) samples, the culture medium was collected promptly upon addition of His-apelin-55; thus, exposure to His-apelin-55 was short (≤ ~ 1 min). For all other treatments, the culture medium was collected at the designated incubation periods. All treatments were carried out in triplicate and all experiments were at least in duplicate. A high inhibitor treatment (25 μM) was used as a control for all inhibitor experiments.
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