The hippocampal mouse cells (mHippoE-18, CELLutions Biosystems, Ontario, Canada) were routinely maintained in DMEM high-glucose culture medium (Sigma-Aldrich, Missouri, MO, USA) without sodium pyruvate. The THP1-Blue™ NF-κB cells derived from the human monocytic THP-1 cell line (Invivogen, Toulouse, France) were maintained in RPMI 1640 medium (Cytogen, Zgierz, Poland). Media for both cell lines were supplemented with 10% fetal bovine serum (Biowest, Nuaille, France), 100 U/mL penicillin, and 100 μg/mL streptomycin (Penicillin-Streptomycin Solution ATCC®). In addition, the medium for THP1-Blue™ NF-κB was supplemented with 100 μg/mL normocin (InvivoGen, San Diego, CA, USA), and 10 μg/mL blastocidin (InvivoGen, USA). The mHippoE-18 cells were trypsinized (Trypsin-EDTA Solution, ATCC®) twice a week, seeded at a density of 5 × 105 cells per T25 cell culture flask, and incubated at 37 °C and 5% CO2 to obtain a confluent monolayer. The THP1-Blue™ NF-κB cells were also incubated at 37 °C in a humidified incubator with 5% CO2 and passaged every 3 days to maintain density < 2 × 106 cells/mL.
High glucose dmem culture medium
Manufactured by Merck Group
Sourced in United States
High glucose DMEM culture medium is a cell culture medium developed by Merck Group. It contains a higher concentration of glucose compared to standard DMEM formulations, providing a nutrient-rich environment for the growth and maintenance of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Poly l ornithine, by Merck Group (2 mentions)
Collagenase d, by Roche (2 mentions)
Trypsin trl, by Worthington (2 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Penicillin streptomycin solution, by American Type Culture Collection (1 mentions)
Normocin, by InvivoGen (1 mentions)
Thp1 blue nf κb cells, by InvivoGen (1 mentions)
Blastocidin, by InvivoGen (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Octanoic acid, by Merck Group (1 mentions)
Sebacic acid, by Merck Group (1 mentions)
Decanoic acid, by Merck Group (1 mentions)
T0070907, by Bio-Techne (1 mentions)
Gsk3787, by Bio-Techne (1 mentions)
Ciglitazone, by Bio-Techne (1 mentions)
Gw6471, by Bio-Techne (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
5 protocols using high glucose dmem culture medium
1
Culturing Mouse Hippocampal and Human Monocytic Cells
2
Huh7 Cell Culture and Treatments
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Huh7 (Japanese Collection of Research Bioresources Cell Bank, no. JCRB0403, Japan) cells were cultured in DMEM high glucose culture medium (Sigma‐Aldrich Co. LLC. no. D5796) supplemented with 10% FBS (Invitrogen), 1× penicillin/streptomycin (Sigma) and non‐essential amino acids (Sigma) in Normoxic conditions (37°C, 5% CO2). Cells were passaged at 70–80% confluency using 0.05% Trypsin in PBS (Severn Biotech). Cells were used experimentally up to passage 10. For treatment, cells were seeded into 6‐well plates at 2 × 105 cells per well and allowed to recover for 48 h. Treatment compounds were added directly into culture medium. Cells were treated for 4 h either under standard conditions or in stress conditions (2% O2, 10% CO2, 32°C; combined hypoxia, hypercapnia and hypothermia) with a vehicle control (DMSO unless otherwise indicated) or compound of interest: 2‐ene‐VPA (2VPA; MolPort), 2‐propyloctanoic acid (2POA; Sigma), ciglitazone (Tocris), decanoic acid (Sigma), GSK3787 (Tocris), GW6471 (Tocris), octanoic acid (Sigma), sebacic acid (SA; Sigma), T0070907 (Tocris), VPA (Sigma, vehicle dH2O), valpromide (VPD; Katwijk Chemie, The Netherlands).
3
Dorsal Root Ganglia Isolation and Culture
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DRGs from mice and rats were harvested below vertebral levels T9 and T10, respectively, down to L6 in each case. Ganglia were surgically desheathed before being transferred in high glucose DMEM culture medium (Sigma-Aldrich, St Louis, MO) containing trypsin TRL (0.3 mg/ml, Worthington Biochemical Corporation, Lakewood, NJ) and collagenase D (1.4 mg/ml, Roche Life Science, Penzberg, Germany). After 40 min incubation under constant shaking at 34 °C, digested DRG fragments were washed by two successive centrifugations and triturated with a fire-polished glass Pasteur pipette. Cells were plated on 8 mm glass coverslips coated with poly-L-ornithine (Sigma-Aldrich, St Louis, MO) in DMEM without serum or growth factors, and incubated overnight at 37 °C, 5% CO2 and 95% humidity.
4
Culturing HNC Cell Lines FADU and HN13
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HNC cancer cell lines FADU (pharyngeal cancer) and HN13 (cancer of the oral cavity) were thawed and cultured in 25 cm2 and 75 cm2 flasks containing high glucose DMEM culture medium (Sigma, San Louis, MO, USA), supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 units/mL of sodium penicillin, 100 µg/mL of streptomycin (Invitrogen, Waltham, MA, USA) and 1% L-glutamine (Gibco, Grand Island, NY, USA). Flasks were kept at 37 °C in an 5% CO2 atmosphere. Media was replaced every three days. Cells were trypsinized when 80% confluence was reached, and the number of cells needed to carry out the transfection was obtained in the second passage for the FADU cell line and in the first passage for the HN13 cell line. Shortly after, cells were washed twice with 1x PBS and treated with Trypsin/EDTA (0.125%/0.05%). Trypsinization was interrupted by the addition of Complete Culture Medium. Cells were then split into new flasks to be kept or to be used in further experiments.
5
Dissociated DRG Neuron Preparation for Nociceptor Analysis
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A dissociated DRG neuron preparation was used that we have repeatedly found in nociceptors from rats, mice, and humans to retain injury-induced, pain-related hyperexcitability for more than 24 hours in culture after in vivo induction of persistent neuropathic pain 8 (link)–11 (link), 42 (link), 52 (link), 53 (link), 81 (link), 84 (link). Rats were euthanized using intraperitoneal injection of pentobarbital/phenytoin solution (Euthasol, 0.9 ml, Virbac AH, Inc.) followed by transcardial perfusion of ice-cold phosphate buffered saline (PBS, Sigma-Aldrich). DRGs were harvested from below the vertebral T10 level to L6. Ganglia were surgically desheathed before transfer to high-glucose DMEM culture medium (Sigma-Aldrich) containing trypsin TRL (0.3 mg/ml, Worthington Biochemical Corporation) and collagenase D (1.4 mg/ml, Roche Life Science). After 40-min incubation under constant shaking at 34°C, digested DRGs were washed by two successive centrifugations and triturated with two fire-polished glass Pasteur pipettes of decreasing diameters. Cells were plated on 8-mm glass coverslips (Warner Instruments) coated with poly-L-ornithine (Sigma-Aldrich) 0.01% in DMEM without serum or growth factors, and incubated overnight at 37°C, 5% CO2 and 95% humidity.
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