Our in vitro studies used murine NIH 3T3 fibroblasts (CRL-1658; American Type Culture Collection, Manassas, VA) in a model system of chronic hyperglycemic (HG) conditions using 25 mmol/L d -glucose DMEM (Life Technologies, Grand Island, NY) versus normoglycemic (NG) conditions using 5 mmol/L d -glucose DMEM (Life Technologies) based on previously published models of hyperglycemia (16 (link)). Media was supplemented with 10% FBS (Life Technologies) and 1% penicillin/streptomycin. Cells were cultured in 37°C at 5% CO2.
D glucose dmem
Manufactured by Thermo Fisher Scientific
Sourced in United States
D-glucose DMEM is a cell culture medium formulation that contains D-glucose as the primary carbohydrate source. It is designed to support the growth and maintenance of a variety of cell lines in vitro. The medium provides a balanced salt solution, amino acids, vitamins, and other essential components necessary for cell proliferation and survival.
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Lab products found in correlation
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Ha tag antibodies, by Merck Group (1 mentions)
Jetprime reagent, by Polyplus Transfection (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Crl 1658, by American Type Culture Collection (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Foetal calf serum, by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Hacat, by Thermo Fisher Scientific (1 mentions)
Cck 8, by Molecular Devices (1 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Atcc htb 96, by American Type Culture Collection (1 mentions)
Penicillin streptomycin cocktail, by Merck Group (1 mentions)
Affinipure bovine anti goat igg, by Jackson ImmunoResearch (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
8 protocols using d glucose dmem
1
In Vitro Hyperglycemic Fibroblast Model
2
Glucose Concentration Influences Cell Behavior
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Experiments groups were classified according to the glucose concentration in the medium.
(1) Low-glucose group (n = 6): 1-g/L D-Glucose DMEM (Gibco 11885-084, Life Technologies)
(2) High-glucose group (n = 6): 4.5-g/L D-Glucose DMEM (Gibco 11965-092, Life Technologies)
(1) Low-glucose group (n = 6): 1-g/L D-Glucose DMEM (Gibco 11885-084, Life Technologies)
(2) High-glucose group (n = 6): 4.5-g/L D-Glucose DMEM (Gibco 11965-092, Life Technologies)
3
Cell Culture of HEK293T, HEK293, and Jurkat
4
Recombinant IDS Construct Generation
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IDS WT and IDS W337X constructs were ordered by Gen-Script (Piscataway). IDS R443X , IDS Y452X , and IDS L482X were generated in our laboratory, amplifying pcDNA3.1 containing the IDS WT sequence by PCR using 5¢-GATCACA CCGGTACCCCACCATGCCGCCA-3¢ as forward primer and, as reverse primers, 5¢-ACCTTCCTGAAGCATTTTG CGGCCGCCCCACACTAG-3¢ for IDS R443X , 5¢-ACTT GGAAGAGGATCCGGCGGCCGCCCCACACTAG-3¢ for IDS Y452X , and 5¢-ATTCTGACAAGCCGAGTGCGGCCG CCCCACACTAG-3¢ for IDS L482X . HA-tag antibodies are from Sigma-Aldrich (Buchs, Switzerland), anti-IDS from R&D Systems (Minneapolis). PS341 (Millenium Pharmaceuticals), and BafA1 (Calbiochem and Sigma Aldrich, Buchs, Switzerland) were used at final concentration of 10 mM and 50-100 nM, respectively.
Cell lines and transient transfection HEK and mouse embryonic fibroblasts (MEF) were cultured in 4.5 g/L D glucose D-MEM (Thermo Fisher Scientific, Waltham) containing 10% fetal bovine serum. Cells were seeded in 6 cm culture dishes/12-well plates. Transfection was performed using JetPrime reagent (Polyplus transfection, Illkirch, France) and 1-1.5 mg of total IDS plasmid. Experiments were performed 17 h post-transfections.
Cell lines and transient transfection HEK and mouse embryonic fibroblasts (MEF) were cultured in 4.5 g/L D glucose D-MEM (Thermo Fisher Scientific, Waltham) containing 10% fetal bovine serum. Cells were seeded in 6 cm culture dishes/12-well plates. Transfection was performed using JetPrime reagent (Polyplus transfection, Illkirch, France) and 1-1.5 mg of total IDS plasmid. Experiments were performed 17 h post-transfections.
5
Investigating PFN1 Function in Diabetic Nephropathy
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To investigate the function of PFN1 in DN, the PFN1 gene was overexpressed or knocked out. PFN1 shRNA and PFN1 overexpression vectors were constructed (Table S1
HK-2 cells of passage 3 to 12 (P3-P12) were inoculated into 6-well plates (approximately 2×105 cells per well). Cells were divided into six groups, including the control group [normal glucose (NG), 5.5 mmol/L D-glucose], the high glucose (HG) induced for 72 h group (45 mmol/L D-glucose), the shRNA vector (SV) transfection + HG induced for 72 h group, the PFN1 shRNA (SR) transfection + HG induced for 72 h group, the plasmid vector (PV) transfection + HG induced for 72 h group, and the PFN1 plasmid transfection + HG induced for 72 h group. Cells were transfected with shRNA or plasmid using Lipofectamine 2000 (Invitrogen, USA). After 24 h of transfection, cells were replaced with high glucose medium (45 mmol/L D-glucose DMEM (Gibco, USA) + 10%FBS).
HK-2 cells of passage 3 to 12 (P3-P12) were inoculated into 6-well plates (approximately 2×105 cells per well). Cells were divided into six groups, including the control group [normal glucose (NG), 5.5 mmol/L D-glucose], the high glucose (HG) induced for 72 h group (45 mmol/L D-glucose), the shRNA vector (SV) transfection + HG induced for 72 h group, the PFN1 shRNA (SR) transfection + HG induced for 72 h group, the plasmid vector (PV) transfection + HG induced for 72 h group, and the PFN1 plasmid transfection + HG induced for 72 h group. Cells were transfected with shRNA or plasmid using Lipofectamine 2000 (Invitrogen, USA). After 24 h of transfection, cells were replaced with high glucose medium (45 mmol/L D-glucose DMEM (Gibco, USA) + 10%FBS).
6
Human Mesangial Cell Culture and Stimulation
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Cultures of human mesangial cells were supported kindly by Professor Shiyonghong from Hebei Medical University. The cells were maintained in a special media supplemented with 1 g/L D-glucose DMEM (GIBCO), 5%–10% foetal calf serum (GIBCO), and 1% Pen-Strep (GIBCO). Cells in the third to seventh passages were used in all in vitro experiments. Depending on different experiments, HMCs were pretreated with a serum-free medium for 24 h and then stimulated with DMEM (control), Aldo (1 μM), aldosterone with eplerenone (10 μM), and aldosterone with serum containing the HJHR at indicated time points.
7
In Vitro Evaluation of Keratinocyte Response to Hyperglycemia and Therapeutic Agents
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Human immortalized keratinocytes (HaCaT) purchased from National Infrastructure of Cell Line Resource was used for our in vitro experiment. Briefly, HaCaT cells were cultured with culture medium which contained DMEM (Gibco, USA) with D-glucose (1 g/L, LG), fetal bovine serum (10%v/v, FBS), and penicillin/streptomycin (100 μg/mL) at 37°C, and stimulation of HaCaT cells with culture medium containing 4.5 g/L D-glucose DMEM (HG) for 48 hours was used for chronic hyperglycemic cell model. Povidone iodine and quercetin were purchased from Hubei Ketian Pharmaceutical Co., Ltd and National Institutes for Food and Drug Control, respectively. First, HaCaT cells were treated with various concentrations of HB, quercetin, and povidone iodine, respectively, for 48 hours to search the safe concentration. To evaluate the therapeutic effect, cells were treated with HG medium and drugs such as HB (1/50 and 1/5000, dilution), quercetin (1.5625 and 6.25 μM), and povidone iodine (0.00001% and 0.001%, m/v) at the same time for 48 hours before detection. CCK-8 was used for detecting cell viability with a microplate reader (Molecular Devices, USA).
8
Culturing Human and Mouse Cell Lines
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Human osteosarcoma cells (U2OS, ATCC ® HTB-96 ™ ), mouse embryonic fibroblasts (MEFs) with/without S51A mutation of eIF2𝛼, HAP1 cells: (a) parental, (b) eIF2a (S51A), (c) DHRI, (d) DGCN2, (e) DPKR, (f) DPERK (Horizon Discovery, UK) were grown in Dulbecco's Modified Eagle Medium with 4.5 g/L D-glucose (DMEM, Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and Penicillin-Streptomycin cocktail (Sigma-Aldrich). AffiniPure Bovine Anti-Goat IgG (805-605-180) and were purchased from Jackson ImmunoResearch.
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