Dmem growth media

Manufactured by Thermo Fisher Scientific

Sourced in United States

DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium used for the growth and maintenance of a wide variety of cell lines. It provides the necessary nutrients and components to support cell proliferation and survival in an in vitro environment.

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9 protocols using dmem growth media

Melanoma and Lung Cancer Cell Lines

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The A375 and Malme-3M melanoma cell lines lines used in this study were purchased from the ATCC. The WM983B melanoma cell line was a gift from L. Larue (Institut Curie, France). The A375 and WM983B cell lines were maintained at 37 °C and 5% CO₂ in a humidified atmosphere and grown in Dulbecco’s modified Eagle’s medium (DMEM) growth media supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine (Gibco). The Malme-3M cells were grown in Iscove’s modified Dulbecco’s medium supplemented with 20% FBS. The PC9 lung cancer cell line was grown in RPMI1640 supplemented with 10% FBS. All cell lines were regularly controlled to be mycoplasma-free by using a PCR-based test (Biovalley). BRAF inhibitor (PLX4032, #S1267), MEKi (Cobimetinib, #S8041), EGFR inhibitor (Erlotinib, #S1023), KDM6B inhibitor (GSK-J1, #S7581) and CBP/p300 inhibitor (SGC-CBP30, #S7256) were purchased from Selleckchem (Euromedex, France). Silvestrol was purchased from MedChem Tronica (# HY-13251, Sweden). Pateamine A was provided by S. Apcher (Gustave Roussy Institute, France). Hippuristanol was provided by J. Tanaka (University of the Ryukyus, Japan). 4E1RCat (#SML0197) and cycloheximide (CHX, #C104450) was purchased from Sigma. All the chemicals were dissolved in dimethylsulfoxide (DMSO) for in vitro studies. The panel of small-molecule chemical library was obtained from L. Désaubry (Strasbourg, France).

Shen S., Faouzi S., Bastide A., Martineau S., Malka-Mahieu H., Fu Y., Sun X., Mateus C., Routier E., Roy S., Desaubry L., André F., Eggermont A., David A., Scoazec J.Y., Vagner S, & Robert C. (2019). An epitranscriptomic mechanism underlies selective mRNA translation remodelling in melanoma persister cells. Nature Communications, 10, 5713.

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Culturing NIH-3T3 Murine Fibroblasts

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NIH-3T3 murine embryonic fibroblasts were purchased from the American Type Culture Collection (ATCC) (VA, USA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) growth media (GIBCO, ThermoFisher Scientific Australia) supplemented with 10% (v/v) foetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin (Life Technologies Australia). All cultures were grown at 37 °C in an atmosphere of 5% CO₂/95% air under aseptic conditions.

Martinovich K.M., Iosifidis T., Buckley A.G., Looi K., Ling K.M., Sutanto E.N., Kicic-Starcevich E., Garratt L.W., Shaw N.C., Montgomery S., Lannigan F.J., Knight D.A., Kicic A, & Stick S.M. (2017). Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics. Scientific Reports, 7, 17971.

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Measurement of Cellular cGMP Levels

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A7r5 rat aortic smooth muscle cells (RAoSM) (ATCC, Rockville, MD, United States) were cultured in 48-well clusters in DMEM growth media (GibcoTM), supplemented with 10% Fetal Bovine Serum (GibcoTM) and 1% Penicillin/Streptomycin (Biosera).
At confluence, cells were serum-starved for 2 h in media containing 0.1% BSA and were then pre-exposed for 5 min to the non-selective phosphodiesterase (PDE) inhibitor isobutyl-methyl-xanthine (IBMX, 1 mM). Then, they were treated with the sGC stimulator BAY 41-2272 at 10 μΜ or a combination of the heme-dependent NO donor sodium nitroprusside (SNP at 100 μΜ) and BAY 41-2272, for 15 min. Some of the cells that received the BAY compound or the BAY/SNP combination were also treated with the heme-oxidizing compound 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (at 10 μΜ) or its vehicle control (DMSO), either 20 min before the addition of IBMX (and hence before the sGC agonists) or 2 min after the addition of the agonist(s).
Cellular cGMP content was assessed as previously described (Argyriou et al., 2021 (link)), by collecting the extracts with HCl 0.1 M, which were analyzed by a commercial ELISA kit according to the manufacturer’s instructions. The cGMP levels in each well were normalized for the respective total protein determined by a Micro BCA Protein assay kit.

Makrynitsa G.I., Argyriou A.I., Zompra A.A., Salagiannis K., Vazoura V., Papapetropoulos A., Topouzis S, & Spyroulias G.A. (2022). Mapping of the sGC Stimulator BAY 41-2272 Binding Site on H-NOX Domain and Its Regulation by the Redox State of the Heme. Frontiers in Cell and Developmental Biology, 10, 925457.

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Athlete Serum Effects on Adipocyte and Myoblast

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Mouse 3T3-L1 preadipocytes (CL-173, ATCC, Manassas, United States) and C2C12 myoblast cell line (CRL-1772, ATCC, Manassas, United States) were cultured in DMEM growth media (Gibco, Thermo Fisher Scientific, Waltham, United States) supplemented with 10% heat-inactivated bovine calf serum (Sigma-Aldrich, Darmstadt, Germany), 1% antibiotics (Gibco), and 1% L-glutamine (Gibco). After reaching confluency, cells were seeded in a 24-well plate at a seeding density of 42, 0000/well. After 24 h, the medium was changed to DMEM growth media without serum for 24 h. Following cell’s starvation, cells were washed once with phosphate buffered saline (PBS) to remove any remnants of FCS and then treated with a conditioned media containing 10% human serum of one of the different athlete serum groups (LE/LP, HE, and HP) or the non-athlete human serum (n = 3 per group) as a negative control. Conventional fetal bovine serum (FBS)-containing media was also used as a control for cell culture. After 72 h incubation, media supernatants were collected from all wells for measurement of cytokines, and the cells were lysed using RIPA buffer (Thermo Fisher Scientific) to measure insulin signaling proteins’ phosphorylation.

Alheidous S., Al-Muraikhy S., Rizk N., Sellami M., Donati F., Botre F., Al-Mansoori L, & Elrayess M.A. (2022). Effect of sera from elite athletes on cytokine secretion and insulin signaling in preadipocytes and skeletal muscle cells. Frontiers in Molecular Biosciences, 9, 943034.

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Maintenance of Cell Lines under Hypoxia

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Cell lines were maintained at 37 °C in a humidified atmosphere at 5% CO2 and grown in RPMI 1640 or DMEM growth media (Invitrogen) supplemented with 10% fetal bovine serum (Sigma), 50 units ml⁻¹ penicillin and 50 μg ml⁻¹ streptomycin (Invitrogen). The following cell lines were maintained in RPMI 1640: SK23, MEL501, MEL526, MEL624. The following cell lines were maintained in DMEM: BJ, IMR90, MEL103, MEL187, RPMI 8322, VMM39, WM2664. Hypoxic conditions (1% O2) were achieved in a Ruskinn in-vivo2 400 hypoxia chamber, by supplementing ambient air with balanced N₂ and CO₂.

Lakhter A.J., Hamilton J., Dagher P.C., Mukkamala S., Hato T., Dong X.C., Mayo L.D., Harris R.A., Shekhar A., Ivan M., Brustovetsky N, & Naidu S.R. (2014). Ferroxitosis: A cell death from modulation of oxidative phosphorylation and PKM2-dependent glycolysis in melanoma. Oncotarget, 5(24), 12694-12703.

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Defining Key Amino Acids of Ro-BatCoV GCCDC1 p10 Protein

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As the p10 protein of Ro-BatCoV GCCDC1 is the first reported in an enveloped virus, we first tried to define the key amino acids for the p10 protein in the Ro-BatCoV GCCDC1 as described previously for p10 protein of reoviruses [24 (link), 25 (link)]. For syncytial indexing of six mutant constructs, each well of BHK-21 monolayer cells in a 6-well plate were transfected with 2 μg of plasmid DNA using Polyethylimine (PEI, Polysciences Inc.) and incubated for 5 h before replacing the transfection mixture with DMEM growth media (Invitrogen) supplemented with 10% fetal bovine serum (GIBCO). Transfected cells were paraformaldehyde-fixed and stained with Wright-Giemsa at the indicated times, and syncytia were observed and pictures were taken at ×100 magnification on an Olympus IX51FL+DP70 microscope.

Huang C., Liu W.J., Xu W., Jin T., Zhao Y., Song J., Shi Y., Ji W., Jia H., Zhou Y., Wen H., Zhao H., Liu H., Li H., Wang Q., Wu Y., Wang L., Liu D., Liu G., Yu H., Holmes E.C., Lu L, & Gao G.F. (2016). A Bat-Derived Putative Cross-Family Recombinant Coronavirus with a Reovirus Gene. PLoS Pathogens, 12(9), e1005883.

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Comprehensive OAC Cell Line Protocols

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OE33 (Cat. No. 96070808), FLO-1 (Cat. No. 11012001), SKGT4 (Cat. No. 11012007), and OE19 (Cat. No. 96071721) OAC cell lines were obtained from Public Health England. MFD-1 cells were a kind gift from Professor Tim Underwood, University of Southhampton [7 (link)]. OE33, SKGT4, and OE19 cells were maintained in RPMI growth media, while FLO-1 and MFD-1 cells were maintained in DMEM growth media (ThermoFisher, Loughborough, UK, Cat. No 11875093 and 11965092). Growth media was supplemented with 10% foetal bovine serum (FBS) (ThermoFisher Cat. No 10500064), 1% sodium pyruvate (ThermoFisher Cat. No 11360070), and 1% penicillin/streptomycin (ThermoFisher Cat. No 15140122) at 37 °C in a humidified atmosphere containing 5% CO₂. All cell lines were routinely examined for mycoplasma contamination using PlasmoTest^TM (Invivogen, Toulouse, France, Cat. No. rep-pt1).

McCabe N.H., Stevenson L., Scanlon E., Douglas R., Kennedy S., Keminer O., Windshügel B., Zisterer D., Kennedy R.D., Blayney J.K, & Turkington R.C. (2022). Identification of Src as a Therapeutic Target in Oesophageal Adenocarcinoma through Functional Genomic and High-Throughput Drug Screening Approaches. Cancers, 14(15), 3726.

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Cell Viability Assay of Algae Extracts

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Monolayers of HeLa cells (ATCC # CCL-2) were grown in culture dishes in DMEM growth media (Thermo Fisher Scientific), 5% FBS (Gibco, Thermo Fisher Scientific) supplemented with 1 mM pyruvate (Thermo Fisher Scientific), 2 mM glutamine (Thermo Fisher Scientific), and 100 IU/ml penicillin/streptomycin (Thermo Fisher Scientific) at 37°C and 5% CO₂. Vero cells (ATCC # CCL-81) were grown in similarly with DMEM as described above. Primary cultures of human gingival fibroblasts derived from the tissue of the masticatory mucosa in the retromolar area of healthy adults (Institutional Ethical Committee approval #60823025) were generated using the explant method and grown in RPMI growth media (Thermo Fisher Scientific; Häkkinen and Larjava, 1992 (link); Arancibia et al., 2009 (link)). Cell viability was determined for HeLa cells and gingival fibroblasts in black wall 96-well clear-bottom plates treated with algae extracts at increasing concentrations for 24 h and then incubated with alamarBlue® (resazurin, Thermo Fisher Scientific) at a 1:10 v/v ratio for 1 h at 37°C and measured in a Synergy Neo HTS Multi-Mode Reader (Biotek).

Castillo E., Duarte L.F., Corrales N., Álvarez D.M., Farías M.A., Henríquez A., Smith P.C., Agurto-Muñoz C, & González P.A. (2020). Anti-herpetic Activity of Macrocystis pyrifera and Durvillaea antarctica Algae Extracts Against HSV-1 and HSV-2. Frontiers in Microbiology, 11, 2006.

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Culturing and Cryopreserving K562 and SK-N-BE(2) Cells

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K562 (ATCC® CCL-243™) and SK-N-BE(2) (ATCC® CRL-2271™) cell lines were acquired from ATCC. All cell lines were tested for mycoplasma every 4–6 months using Alfa Aesar J66117 PCR Mycoplasma Detection Kit; only mycoplasma negative cells were utilized. K562 cells were cultured in suspension with RPMI GlutaMAX media containing 10% fetal bovine serum and 1% Anti-Anti antibiotic/antimycotic (Thermo Fisher Scientific, Waltham, MA) to a density of 1.5 × 10⁶ cells/mL. SK-N-BE(2) cells were cultured on tissue culture treated plates in DMEM growth media (ThermoFisher, 11965118) supplemented with 10% fetal bovine serum, 2 mM l-Glutamine (ThermoFisher, 35050079), and 1% Anti-Anti antibiotic/antimycotic to 70% confluence. In all, 50 million cells were washed two times with phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO₄, and 1.8 mM KH₂PO₄, pH 7.4) and snap frozen in liquid nitrogen for use in-lysate experiments or were used immediately for in-cell experiments.

Ball K.A., Webb K.J., Coleman S.J., Cozzolino K.A., Jacobsen J., Jones K.R., Stowell M.H, & Old W.M. (2020). An isothermal shift assay for proteome scale drug-target identification. Communications Biology, 3, 75.

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