Dulbecco s modified essential medium dmem

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Dulbecco's modified essential medium (DMEM) is a cell culture medium commonly used to support the growth of various cell types in vitro. It provides a balanced salt solution, amino acids, vitamins, and other nutrients required for cell maintenance and proliferation.

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7 protocols using dulbecco s modified essential medium dmem

Propagation of Infectious Bronchitis Virus

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All IBV stocks were propagated in 10-day-old specific pathogen-free (SPF) Rhode Island Red (RIR) embryonated hens’ eggs. M41-CK (GenBank accession number MK728875.1) is a pathogenic IBV belonging to the GI-1 genotype and Massachusetts serotype that has been adapted for propagation in primary chicken kidney (CK) cells [40, 42, 43 (link)] through serial passage in CK cells. Beau-R [41 (link)] is a molecular clone of Beau-CK, the CK cell-adapted Beaudette strain (GenBank accession number AJ311317) also belonging to the GI-1 genotype and Massachusetts serotype. Unlike M41-CK, Beau-R exhibits an extended host tropism, allowing propagation in DF-1 cells [6, 44 (link)].
CK cells were prepared using kidneys from 2- to 3-week-old SPF RIR chickens by the central services unit at The Pirbright Institute [45 (link)]. DF-1 cells were obtained from Central Services Unit at The Pirbright Institute and maintained in Dulbecco’s modified essential medium (DMEM, Sigma-Aldrich, St Louis, MO, USA) with 10 % foetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO, USA). All cells were maintained at 37 °C in 5 % CO₂.

Stevenson-Leggett P., Armstrong S., Keep S., Britton P, & Bickerton E. (2021). Analysis of the avian coronavirus spike protein reveals heterogeneity in the glycans present. The Journal of General Virology, 102(8), 001642.

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Isolation and Expansion of Adipose-Derived Stromal Cells

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Fat tissues were obtained from subcutaneous adipose tissues derived from aesthetic liposuctions. As described previously,^{14 (link)} after 5 times of washing steps with phosphate buffered saline (PBS; Sigma-Aldrich, St. Louis, USA) the tissue samples were incubated at 37°C for 45 min in Dulbecco’s modified essential medium (DMEM; Sigma-Aldrich, St. Louis, USA) containing 2 mg/mL of collagenase (Worthington Biochemical Corp., Lakewood, NJ, USA) with shaking at 60 cycles/min. After centrifugation, the lipid layer was thrown away and the SVF was collected, washed and resuspended in DMEM supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 100 units/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, USA) and seeded into the tissue culture flask (Orange Scientific, Braine-l’Alleud, Belgium). Twenty four-hour post-seeding, the non-adherent cells were discarded by changing the medium, and the cells were harvested and expanded at 50% confluency.

Amirkhani M.A., Mohseni R., Soleimani M., Shoae-Hassani A, & Nilforoushzadeh M.A. (2016). A rapid sonication based method for preparation of stromal vascular fraction and mesenchymal stem cells from fat tissue. BioImpacts : BI, 6(2), 99-104.

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BVDV-1, BVDV-2, and HoBi-like Virus Immunization

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The BVDV-1 NADL (courtesy of Dr. Ferrari, Istituto Zooprofilattico Sperimentale di Lombardia ed. Emilia Romagna, Brescia, Italy), BVDV-2232/02 [17 ], and HoBi-like Pestivirus Italy-83/10cp [21 (link)] cytopathogenic strains were used for immunization procedures (as inactivated preparations) and for the virus neutralization (VN) assays to assess the VN titer in immunized animals. All viruses were propagated in pestivirus-free Madin-Darby bovine kidney cell lines (MDBK; ATCC CCL-22), cultured in Dulbecco’s modified essential medium (DMEM) (Sigma-Aldrich, Schnelldorf, Germany) containing 10% fetal bovine serum (FBS, Gibco, Thermo Scientific, Rodano, Italy, tested negative for pestivirus or anti-BVDV antibody), 2 mM of L-glutamine, 100 IU/ml of penicillin (Sigma-Aldrich), 100 mg/ml of streptomycin (Sigma-Aldrich), and 2.5 mg/ml of amphotericin B. Cells were incubated at 37 °C with 5% CO₂. Human embryo kidney cell lines (HEK293T; ATCC, CRL-1573) were cultured following the same procedure and used for transfection experiments.
For immunization, viral suspensions were inactivated with 0.05% β-propiolactone and emulsified initially with Tween-80 (4.1% v/v) and then with a mixture (50% v/v) of Montanide ISA 563 (Seppic Inc., Paris, France), Marcol 52 (Esso Italiana S.r.l., Rome, Italy), and Montane 80 (Seppic Inc.) in a 30:63:7 ratio, as previously described [22 (link)].

Nogarol C., Decaro N., Bertolotti L., Colitti B., Iotti B., Petrini S., Lucente M.S., Elia G., Perona G., Profiti M., Buonavoglia C, & Rosati S. (2017). Pestivirus infection in cattle dairy farms: E2 glycoprotein ELISA reveals the presence of bovine viral diarrhea virus type 2 in northwestern Italy. BMC Veterinary Research, 13, 377.

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Gadolinium-Based Nanoparticle Synthesis and Functionalization

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Gadolinium(III) chloride hexahydrate (99.9%), sodium hydroxide (>99.9%), triethylene glycol (TEG) (99%), PAA (M_w = ~1800 amu), dimethyl sulfoxide (DMSO) (99.9%), N,N′-dicyclohexylcarbodiimide (DCC) (99%), 4-(dimethylamino) pyridine (DMAP) (>9%), tert-butyl N-(2-aminoethyl) carbamate (EDA-Boc) (>98%), triethylamine (TEA) (>99%), trifluoroacetic acid (TFA) (99%), N-hydroxysuccinimide (NHS) (98%), 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC∙HCl) (97%), FA (>97%), Roswell Park Memorial Institute (RPMI)-1640, Dulbecco’s Modified Essential Medium (DMEM), sterile phosphate-buffered saline (PBS) solution, and dialysis tube (molecular weight cut-off (MWCO) = 1000 and 2000 amu) were procured from Sigma-Aldrich (St. Louis, MO, USA). cRGD (cRGDyk: Arg-Gly-Asp-D-Tyr-Lys) was procured from Vivitide (Gardner, MA, USA). Ethyl acetate (99.9%), chloroform (99.9%), and ethanol (99.99%) were purchased from Duksan (Ansan, Korea). All reagents and materials were used as received. Nanoparticles were initially washed with ethanol, then finally washed with triple-distilled water to prepare nanoparticle suspensions.

Ho S.L., Yue H., Lee S., Tegafaw T., Ahmad M.Y., Liu S., Saidi A.K., Zhao D., Liu Y., Nam S.W., Chae K.S., Chang Y, & Lee G.H. (2022). Mono and Multiple Tumor-Targeting Ligand-Coated Ultrasmall Gadolinium Oxide Nanoparticles: Enhanced Tumor Imaging and Blood Circulation. Pharmaceutics, 14(7), 1458.

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Evaluating Breast Cancer Cell Invasion

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As a first experiment, cancer cells from the highly invasive MDA-MB-231 breast cancer cell line were seeded in 35 mm diameter Petri dishes on glass, tissue-culture polystyrene (TCPS), and type I collagen hydrogels polymerized at 4 mg/ml and temperatures of 4 and 37°C. The gel polymerized at low temperature produced a coarse collagen network (CCN) with larger fiber and pore diameters than the fine collagen network (FCN) produced at 37°C. The MDA-MB-231 cell line was a generous gift from Dr. Zaver Bhujwalla (Johns Hopkins School of Medicine, Baltimore, MD). The cells on collagen were incubated for 24 hours in Dulbecco’s modified essential medium (DMEM, Sigma-Aldrich) containing 10% fetal bovine serum (FBS, Corning), 100 units/ml penicillin, and 100 μg/ml streptomycin (Corning) in standard tissue culture conditions of 37°C, 5% CO₂, and 100% humidity.

Lam V.K., Nguyen T.C., Chung B.M., Nehmetallah G, & Raub C.B. (2017). Quantitative Assessment of Cancer Cell Morphology and Motility Using Telecentric Digital Holographic Microscopy and Machine Learning. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 93(3), 334-345.

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Virus Infection and Titration in CCB Cells

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Triplicate cultures of CCB cells were infected at a multiplicity of infection (MOI) of 0.05 plaque-forming unit (PFU)/cell. Viral inoculums were back-titrated to ensure that the cells were infected with the expected number of PFU. After an incubation period of 2 h, the cells were washed with PBS (the virus recovered in the PBS is reported as time 0) and overlaid with Dulbecco’s modified essential medium (DMEM; Sigma) containing 4.5 g glucose/l and 10 per cent (v/v) FCS. The supernatant was removed from the infected cultures at successive intervals and stored at −80°C. Infectious virions were titrated by duplicate plaque assay in CCB cells as described previously (Ouyang et al. 2013 ).

Gao Y., Sridhar A., Bernard N., He B., Zhang H., Pirotte S., Desmecht S., Vancsok C., Boutier M., Suárez N.M., Davison A.J., Donohoe O, & Vanderplasschen A.F. (2023). Virus-induced interference as a means for accelerating fitness-based selection of cyprinid herpesvirus 3 single-nucleotide variants in vitro and in vivo. Virus Evolution, 9(1), vead003.

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Culturing Human Mesenchymal Stem Cells on Titanium

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Stro-1 + selected primary human MSCs from adult human bone marrow, obtained with informed consent, were provided by Southampton General Hospital (LREC194/99/1 and 18/NW/0231 and 210/01). MSCs were cultured in basal media, (Dulbecco's modified essential medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS; Sigma), 1% (v/v) L glutamine (200 mM, Gibco), 1% sodium pyruvate (11 mg/mL, Sigma), 1% MEM NEAA (amino acids, Gibco), and 2% antibiotics (6.74 U/mL penicillinstreptomycin, 0.2 μg/mL fungizone; Sigma). MSCs were cultured in an incubator set at 37 °C with 5% CO2 environment and sub-cultured to passage 2-3 before use. Culture media was changed every 3 days. Seeding on coated Ti was done in serum-free medium in 24-well plates at 10 4 cells/disc. After 2 hr, the media were changed to low serum growth medium (1% FBS).

Damiati L.A., Tsimbouri M.P., Hernandez V.L., Jayawarna V., Ginty M., Childs P., Xiao Y., Burgess K., Wells J., Sprott M.R., Meek R.M.D., Li P., Oreffo R.O.C., Nobbs A., Ramage G., Su B., Salmeron-Sanchez M, & Dalby M.J. (2022). Materials-driven fibronectin assembly on nanoscale topography enhances mesenchymal stem cell adhesion, protecting cells from bacterial virulence factors and preventing biofilm formation. Biomaterials, 280.

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