HEK293T cells were maintained in Dulbecco's modified Eagle's medium (high glucose) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone), 100 U/ml penicillin and 100 pg/ml streptomycin. Transfections were performed using Lipofectamine 3000 (Life Technologies) or PolyJet™ DNA Tranfection Reagent (SignaGen) according to the manufacturers’ instructions.
High glucose dulbecco s modified eagle s medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, United Kingdom
High-glucose Dulbecco's modified Eagle's medium is a cell culture medium that provides nutrients and growth factors necessary for the maintenance and proliferation of various cell types in vitro. It contains a high concentration of glucose as the primary energy source.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (52 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (20 mentions)
Penicillin, by Thermo Fisher Scientific (12 mentions)
Streptomycin, by Thermo Fisher Scientific (11 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (7 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (3 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Horse serum, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (3 mentions)
Hepes, by Thermo Fisher Scientific (3 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Heat inactivated fetal bovine serum, by Thermo Fisher Scientific (3 mentions)
130 protocols using high glucose dulbecco s modified eagle s medium
1
HEK293T Cell Culture and Transfection
2
Establishing PC-12 and Astrocyte Cell Cultures
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PC-12 cells were from the American Type Culture Collection (CRL-1721) and subjected to quality control tests by the American Type Culture Collection. PC-12 cells were cultured in Dulbecco's modified Eagle's medium (high glucose; Gibco) containing 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 5% heat-inactivated horse serum (HyClone), penicillin (100 units/ml), and streptomycin (1 mg/ml) in plates coated with 10 μg/ml type IV collagen (Sigma–Aldrich). Cells were passaged no more than eight times.
Astrocytes were isolated from Sprague–Dawley rat pup brains, as previously described (67 (link)). In brief, cortices were dissected from the forebrain and surrounding meninges and then mechanically and enzymatically dissociated using the Neural Tissue Dissociation Kit P (Miltenyi Biotec). Mixed glial cultures were established in Dulbecco's modified Eagle's medium/F-12 medium supplemented with GlutaMAX (Gibco), 10% FBS, and 100 units/ml antibiotic–antimycotic (Gibco). After culturing for 10 to 14 days, microglia and oligodendrocytes were removed by shaking. The astrocytes were collected by trypsinization and replated at 3.5 × 105 cells/well on poly-d -lysine-coated surfaces. Experiments were performed within 48 h of completing the isolation procedure.
Astrocytes were isolated from Sprague–Dawley rat pup brains, as previously described (67 (link)). In brief, cortices were dissected from the forebrain and surrounding meninges and then mechanically and enzymatically dissociated using the Neural Tissue Dissociation Kit P (Miltenyi Biotec). Mixed glial cultures were established in Dulbecco's modified Eagle's medium/F-12 medium supplemented with GlutaMAX (Gibco), 10% FBS, and 100 units/ml antibiotic–antimycotic (Gibco). After culturing for 10 to 14 days, microglia and oligodendrocytes were removed by shaking. The astrocytes were collected by trypsinization and replated at 3.5 × 105 cells/well on poly-
3
Cell Culture Conditions for Pancreatic Cancer Cell Lines
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The parental Panc-1 (ATCC®, USA) cell line was cultured with Dulbecco’s Modified Eagle’s Medium, High Glucose (GIBCO, USA); the parental AsPC-1 (ATCC®, USA) cell line was cultured with RPMI 1640 Medium (GIBCO, USA). Both media formulations were supplemented with 10% fetal bovine serum (FBS) (GIBCO, USA) and 1% penicillin/streptomycin/amphotericin (PSA) (GIBCO, USA). The media were changed every two days and the cells were incubated in a humidified chamber at 37 °C in 5% CO2.
4
Culturing Human Cell Lines for Research
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HeLa cells (human cervix carcinoma cell line) were cultured in Dulbecco’s modified Eagle’s medium/high glucose (Gibco, Grand Island, NY, USA) medium supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco). KGN cells (human granulosa cell line) were cultured in Dulbecco’s modified Eagle’s medium/F-12 (Gibco) medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. The cells were cultured at 37°C with 5% CO2.
5
Isolation and Culture of Neonatal Rat Cardiomyocytes
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Primary neonatal rat cardiomyocytes (NRCMs) were isolated and cultured as previously reported.7 (link) Briefly, the left ventricles of 1-day-old neonatal Sprague-Dawley rats (Beijing HFK Bioscience) were harvested and digested with 1.25% trypsin (Thermo Fisher Scientific) at room temperature, then incubated with 0.08% collagenase II (Solarbio Life Sciences) at 37°C. Cells were filtered with a cell strainer (70 μm; BD Falcon). The suspension was centrifuged at 1,500 rpm for 10 minutes. After being washed with PBS, the precipitate was resuspended in Dulbecco’s modified Eagle’s medium–high glucose (Gibco) with 15% fetal bovine serum (Gibco) and 0.1 mM bromodeoxyuridine for 48 hours. Finally, NRCMs were cultured in Dulbecco’s modified Eagle’s medium–high glucose supplemented with 20% healthy or CKD serum and 1% (vol/vol) penicillin/streptomycin for 48 hours. To inhibit ODC1, cells were pretreated with 0.5 mM difluoromethylornithine for 24 hours. To block MOMP, cells were pretreated with 10 μM VBIT-4 for 1 hour. To inhibit BAX, cells were pretreated with 2 μM BAI1 (Selleck Chemicals) for 24 hours. To scavenge reactive oxygen species (ROS), cells were pretreated with 5 mM N-acetylcysteine (Sigma-Aldrich) for 1 hour.
6
Thymic Epithelial Cell Culturing and Manipulation
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The cortical thymic epithelial cell line cTEC 1–2 and the medullary thymic epithelial cell line mTEC 3–10 were kindly provided by Prof. Georg Holländer (Department of Biomedicine, University of Basel) and ST4.5 CD4+ CD8+ thymocyte cell line was provided by Dr. Anne Wilson (Ludwig Institute of Cancer Research, Lausanne). All cell lines were cultured in Dulbecco’s modified Eagle’s medium high glucose (Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin sulfate solution (Gibco).
All cell types were continuously cultured in humidified incubators at 37°C containing 21% O2 (normoxia) or 5% O2 (hypoxia) for at least 1 week prior to experimentation.
Cells were γ-irradiated at the indicated doses using a Maintenance Millennium Sample Irradiator containing a 137Cs source at a dose rate of approximately 102 cGy/min. Cells were treated with 1µM staurosporine solution (Cell Signaling Technologies) or 25 µM 2-bromodeoxiuridine (BrdU) and harvested at the indicated time points post-treatment.
All cell types were continuously cultured in humidified incubators at 37°C containing 21% O2 (normoxia) or 5% O2 (hypoxia) for at least 1 week prior to experimentation.
Cells were γ-irradiated at the indicated doses using a Maintenance Millennium Sample Irradiator containing a 137Cs source at a dose rate of approximately 102 cGy/min. Cells were treated with 1µM staurosporine solution (Cell Signaling Technologies) or 25 µM 2-bromodeoxiuridine (BrdU) and harvested at the indicated time points post-treatment.
7
Diverse Cell Culture Protocols for Biomedical Research
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Human umbilical cord blood-derived MSCs were purchased (CEFO, Seoul, South Korea) and cultured according to the manufacturer’s instructions. MSCs were cultured in Dulbecco’s modified Eagle’s medium low glucose (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco BRL) and 1% (v/v) penicillin/streptomycin (Gibco BRL) and were used for the experiments at passage under six. RAW 264.7 cell line was purchased from Korean Cell Line Bank (Seoul, South Korea) and cultured according to the manufacturer’s instructions. RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium high glucose (Gibco BRL) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. PC12 cells were purchased from Paragon Biotech (Baltimore, MD, USA) and cultured in RPMI 1640 (Gibco BRL) supplemented with 7.5% (v/v) FBS, 7.5% (v/v) horse serum (Gibco BRL), and 1% (v/v) penicillin/streptomycin. HUVECs were purchased from Lonza (Walkersville, MD, USA) and cultured in Endothelial Cell Growth Media-2 (Lonza) on tissue culture plates. HUVECs at a passage under seven were used for the experiments.
8
Culturing MDA-MB-231 Breast Cancer Cells
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MDA-MB-231 (human breast cancer cells) cells were purchased from the National Laboratory Cell Resource (China) and were grown in Dulbecco's Modified Eagle's Medium-high glucose (GIBCO, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator with 5% CO2 at 37°C.
9
Isolation of Neonatal Rat Cardiomyocytes
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Neonatal rat cardiomyocytes were prepared from 1–3 day old Sprague Dawley rats by enzymatic digestion. Hearts were excised and placed in sterile PBS solution. After connective tissue and blood were removed, the ventricles were minced and subjected to 8 min enzymatic digestion using serial 0.06% collagenase II and 0.08% pancreatin (both were purchased from Gibco, Thermo Fisher Scientific Inc., Waltham, USA) digestion. Cells were pre-plated on 10 cm petri dishes for 2 h to remove fibroblast cells and then cultured for 24 h in 10% (v/v) fetal bovine serum in Dulbecco’s modified eagle’s medium (high glucose) (Gibco) and 1% penicillin-streptomycin solution (Gibco) at 37°C in a humidified incubator with 5% CO2. After 24 h the media was replaced with 3% fetal bovine serum containing media.
10
Retinal Induction of Human PDLSCs
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Human PDLSC lines were established previously10 (link)11 (link). These cells were cultured in Dulbecco’s modified Eagle’s medium (high glucose; Gibco BRL, Rockville, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL). Cells with passage 3–5 were used for the retinal induction experiments.
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