High glucose dmem

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High-glucose DMEM is a cell culture medium that contains a high concentration of glucose (4.5 g/L). It is commonly used to support the growth and maintenance of a variety of cell types in vitro.

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2 356 protocols using high glucose dmem

Cell Culture Protocols for Cancer Research

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A549 cells: A549 cells derived from two different sources, American Type Culture Collection (ATCC) and China Center for Type Culture Collection (CCTCC). The A549 cells were maintained in DMEM high glucose (Gibco™, USA) with 10% fetal bovine serum (Zeta life, USA) and 1% penicillin/streptomycin (New Cell & Molecular Biotech, Co., Ltd).
HT-29 cells: HT-29 cells were derived from CCTCC. The HT-29 cells were maintained in DMEM high glucose (Gibco^TM, USA) with 10% fetal bovine serum (Zeta life, USA) and 1% penicillin/streptomycin (New Cell & Molecular Biotech, Co., Ltd).
CAL-27 cells: CAL-27 cells were derived from CCTCC. The CAL-27 cells were maintained in DMEM high glucose (Gibco^TM, USA) with 10% fetal bovine serum (Zeta life, USA) and 1% penicillin/streptomycin (New Cell & Molecular Biotech, Co., Ltd).
MDA-MB-231 cells: MDA-MB-231 cells were derived from CCTCC. The MDA-MB-231 cells were maintained in DMEM high glucose (Gibco^TM, USA) with 10% fetal bovine serum (Zeta life, USA).

Tan J., Li H., Ji C., Zhang L., Zhao C., Tang L., Zhang C., Sun Z., Tan W, & Yuan Q. (2022). Electron transfer-triggered imaging of EGFR signaling activity. Nature Communications, 13, 594.

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Generating Stable Cell Lines with Lentiviral Transduction

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Stable cell lines were generated using lentiviruses following a previously established protocol (Andrzejewska et al., 2016 (link)). Briefly, human cystinosin-GFP and its related mutants including N288K, D205N, D305N, D346N were cloned into a lentiviral vector pLVX-EF1a-IRES-Puro. 5 mg of cystinosin plasmid was co-transfected with 2 mg pMD2G and 3 mg pSPAX2 (packaging plasmids) into HEK293T cells using X-tremeGENE HP DNA transfection reagent. After 24 hours, cell medium was changed from high glucose DMEM (Gibco) with 10% FCS, 1% L-glutamine and 1% Penicillin-Streptomycin to high glucose DMEM (Gibco) supplemented with 30% FCS, 1% L-glutamine and 1% Penicillin-Streptomycin at 37 °C with 5% CO₂. The supernatant was collected at 48 hours and 72 hours, and filtered through 0.45-mM pore size filters, yielding 7X lentiviral particles that were ready for use. NIH/3T3 cells were maintained in high glucose DMEM (Gibco) with 10% newborn calf serum and 1% Penicillin-Streptomycin at 37 °C with 8.8% CO₂. To establish NIH/3T3 stable cell lines, cells were infected with 1X lentiviral supernatant in the presence of polybrene (final concentration of 8 mg/ml). At 48 hours post-infection, cells were selected by adding puromycin to a final concentration of 1μg/ml for 2 weeks before the Co-IP assay.

Guo X., Schmiege P., Assafa T.E., Wang R., Xu Y., Donnelly L., Fine M., Ni X., Jiang J., Millhauser G., Feng L, & Li X. (2022). Structure and Mechanism of Human Cystine Exporter Cystinosin. Cell, 185(20), 3739-3752.e18.

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Cell Death Assay in Neuroblastoma Cells

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SY5Y human neuroblastoma cell line, generously donated by Dr. Marion Ehrich VMRCVM, were maintained in L15 media containing 10% FBS. For transient transfection, 60 000 cells were plated per well in a 24-well plate a day before transfection. A complete media change was performed 30 min before transfection, during which time the plasmids pcDNA3.1-V5, pcDNA3.1-V5-EphB3 and pcDNA3.1-V5-EphB3D849N were mixed with Truefect United Biosystems (Herndon, VA, USA) in DMEM/high glucose (Gibco, Langley, OK, USA) according to manufacturer's recommendation and then 25 μl of each complex was added to each respective wells and incubated for 5 h. Media was then removed, washed once with DMEM/high glucose and 500 μl of DMEM/high glucose containing either pre-clusted Fc (0.5 μg/ml) or ephrinB3-Fc (1.0 μg/ml) were added. Forty-eight hours after transfection, trypan blue assay was performed to assess the percentage of cell death for each condition.

Theus M.H., Ricard J., Glass S.J., Travieso L.G, & Liebl D.J. (2014). EphrinB3 blocks EphB3 dependence receptor functions to prevent cell death following traumatic brain injury. Cell Death & Disease, 5(5), e1207-.

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Cell Culture Protocols for Cancer Research

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Human lung carcinoma cell lines A549 and PC9 were obtained from FDCC (Shanghai, China) and maintained in RPMI 1640 medium or DMEM (High Glucose) (Thermo, Suzhou, China) supplemented with 10% FBS (Gemini, USA) and 1% antibiotics (penicillin-streptomycin, Gibco, USA). Human brain cancer cell line U87 were obtained from ATCC (USA) and maintained in DMEM (High Glucose) (Thermo, Suzhou, China) supplemented with 10% FBS (Gemini, USA) and 1% antibiotics (penicillin-streptomycin, Gibco, USA). Human liver cancer cell line HepG2, breast cancer cell line MCF-7, bladder cancer cell line EJ were obtained from SIBS (Shanghai, China) and maintained in RPMI 1640 or DMEM (High Glucose) (Thermo, Suzhou, China) medium supplemented with 10% FBS (Gemini, USA) and 1% antibiotics (penicillin-streptomycin, Gibco, USA). Human retinal pigment epithelium (ARPE-19) cells were bought from iCell Bioscience (Shanghai, China) and maintained in DMEM/F12 cell medium (Hyclone, USA) supplemented with 10% FBS (Gemini, USA) and 1% antibiotics (penicillin-streptomycin, Gibco, USA). MCF-10A were obtained from SIBS (Shanghai, China) and maintained in DMEM (Invitrogen, USA) supplemented with 20% Horse Serum (Invitrogen, USA), 0.02% EGF (Peprotech, USA) , 0.05% Hydrocortisone (Sigma, USA), 0.1%Insulin (Sigma, USA) and 1% antibiotics (penicillin-streptomycin, Gibco, USA). Roscovitine was from Selleck (Shanghai, China).

Zeng J., Xie S., Liu Y., Shen C., Song X., Zhou G.L, & Wang C. (2018). CDK5 Functions as a Tumor Promoter in Human Lung Cancer. Journal of Cancer, 9(21), 3950-3961.

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Cell Culture Protocol for Three Cell Lines

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Three cell lines (HEK293T, SH-SY5Y, and U251) were originally obtained from the Kunming Cell Bank at the Kunming Institute of Zoology, Chinese Academy of Sciences. HEK293T and U251 cells were cultured in high-glucose DMEM (Gibco, Cat. No: C11995500BT) supplemented with 10% FBS (Gibco, Cat. No: 10091148) and 1% penicillin and streptomycin (100 U/mL). SH-SY5Y cells were cultured in high-glucose DMEM (Gibco, Cat. No: C12430500BT) supplemented with 10% FBS (Gibco, Cat. No: 10091148), 10 mM sodium pyruvate solution (Gibco, Cat. No: 11360070), 1% penicillin and streptomycin (100 U/mL), and 1× minimum essential medium nonessential amino acid solution (Gibco, Cat. No: 11140050). All cells were cultured at 37 °C in 5% CO₂. All cell lines were confirmed to be mycoplasma-free by regular testing by PCR analysis.

Chen R., Yang Z., Liu J., Cai X., Huo Y., Zhang Z., Li M., Chang H, & Luo X.J. (2022). Functional genomic analysis delineates regulatory mechanisms of GWAS-identified bipolar disorder risk variants. Genome Medicine, 14, 53.

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Mechanical Wound Healing Scratch Assay

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pDFs obtained from WT, CERK-KO, and cPLA₂α-KI mice were plated at a density of 2 × 10⁶ on 100 mm tissue culture plates in high glucose DMEM supplemented with 10% FBS (Gibco) and 2% penicillin/streptomycin (Bio Whittaker) and left overnight to adhere at standard incubation conditions. Following the overnight incubation, cells were rested in 2% FBS (Gibco), 2% penicillin/streptomycin (Bio Whittaker), and high glucose DMEM (Gibco) for 2 h. After the 2 h resting period, mechanical trauma was induced on the monolayer by performing scratches across the diameter of the plate in an asterisk pattern using four 20 μl pipette tips on a multichannel micropipette. Media were taken for lipidomic analysis at multiple time points (0 h and 2 h).

Maus K.D., Stephenson D.J., Ali A.N., MacKnight H.P., Huang H.J., Serrats J., Kim M., Diegelmann R.F, & Chalfant C.E. (2022). Ceramide kinase regulates acute wound healing by suppressing 5-oxo-ETE biosynthesis and signaling via its receptor OXER1. Journal of Lipid Research, 63(4), 100187.

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Culturing FSHD2 Myoblasts from Patient Biopsies

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Human control and FSHD2 myoblast cells from patient quadricep and tibia biopsies were grown on dishes coated with collagen in high glucose DMEM (Gibco) supplemented with 20% FBS (Omega Scientific, Inc.), 1% Pen-Strep (Gibco), and 2% Ultrasor G (Crescent Chemical Co.) [21 ]. Upon reaching 80% confluence, differentiation was induced by using high glucose DMEM medium supplemented with 2% FBS and ITS supplement (insulin 0.1%, 0.000067% sodium selenite, 0.055% transferrin; Invitrogen). Fresh differentiation medium was changed every 24hrs.

Jiang S., Williams K., Kong X., Zeng W., Nguyen N.V., Ma X., Tawil R., Yokomori K, & Mortazavi A. (2020). Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei. PLoS Genetics, 16(5), e1008754.

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Maintenance of Mouse Embryonic Stem Cells

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mESCs (R1) were kindly provided by Dr Huang Tian Yang (Shanghai Jiao Tong University School of Medicine, Shanghai, China). R1 cells were maintained in high glucose DMEM (Gibco) with 15% fetal serum (Vian-saga), 1000 units leukemia inhibitory factor (LIF) (Millipore, Bedford, MA, U.S.A.), 100 mM β-mercaptoethanol (Sigma), 2 mM non-essential amino acids (Gibco. Inc), 100 units/ml penicillin, and 100 mg/ml streptomycin (Sigma–Aldrich, St. Louis, MO, U.S.A.) without feeder cells. Colonies were dissociated using trypsin and passaged at a 1:3 split ratio every 3 4 days depending on the cell density. 293T cells were maintained in high glucose DMEM (Gibco) with 10% fetal serum (Vian-saga), 100 units/ml penicillin, and 100 mg/ml streptomycin.

Lu Q., Liu Y., Wang Y., Wang W., Yang Z., Li T., Tian Y., Chen P., Ma K., Jia Z, & Zhou C. (2017). Rapamycin efficiently promotes cardiac differentiation of mouse embryonic stem cells. Bioscience Reports, 37(3), BSR20160552.

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Rasal2 Knockdown Adipogenesis Assay

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Cells were maintained in expansion medium [High glucose DMEM (Gibco, 11965118) supplemented with 10% FCS (Gibco, 16170078), 1% Penicillin-Streptomycin (Gibco, 10378016)]. Transfection of siRNA against Rasal2 was performed as previously described [18] (link). In brief, 3T3-L1 preadipocytes were trypsinized, resuspended in expansion medium, then seeded into 24-well plates and transfected with siRNA by Lipofectamine RNAiMAX (Life Technologies). Two days after reaching confluency, cells were induced with a defined adipogenic cocktail [High glucose DMEM (Sigma, 11965118) supplemented with 10% FBS (Gibco, 16000044), 1% Penicillin-Streptomycin (Gibco, 10378016), 5 μg/ml insulin (Sigma, I0259), Dexamethasone (Sigma, D1756), and 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX) (Sigma, I7018)] for 2 days, then raised in the maintenance medium [High glucose DMEM (11965118) supplemented with 10% FBS (Gibco, 16000044), 1% Penicillin-Streptomycin (Gibco, 10378016), and 5 μg/ml insulin (Sigma, I0259)] for additional 4 days. The sequences of Rasal2 siRNA could be found in Table S2.

Zhu X., Xie S., Xu T., Wu X, & Han M. (2017). Rasal2 deficiency reduces adipogenesis and occurrence of obesity-related disorders. Molecular Metabolism, 6(6), 494-502.

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Pancreatic Cell Line Cultivation and Transfection

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The pancreatic epithelial cell lines HPDE6-C7 and PC (PANC-1, CFPAC‑1, and MIAPaCa-2) were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China), and cultured for fewer than 6 months after resuscitation. PANC-1 and HPDE6-C7 cells were maintained in high-glucose DMEM (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% penicillin (Invitrogen, USA). CFPAC1 cells were grown in IMDM (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% penicillin (Invitrogen, USA). MIA-PaCa-2 cells were grown in high -glucose DMEM (Gibco, USA) containing 10% FBS (Gibco, USA), 5% HS (Gibco, USA), and 1% sodium pyruvate and streptomycin (Invitrogen, USA). All cells were cultured in a 5% CO₂ 37 °C humidified incubator.
Cells were transfected with pcDNA-LINC00930, pcDNA-Vector negative control, miR-6792-3p mimic, or miR-NC negative control (RiboBio, China). Lipofectamine® 3000 (Invitrogen, USA; Thermo Fisher Scientific, USA) was used based on provided directions to transfect cells with appropriate constructs.

Mao Y., Su X., Guo Q., Yao X., Zhao Q., Guo Y., Wang Y., Li X, & Lu Y. (2024). Long non-coding RNA LINC00930 targeting miR-6792-3p/ZBTB16 regulates the proliferation and EMT of pancreatic cancer. BMC Cancer, 24, 638.

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