Gibco dulbecco s modified eagle s medium

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Gibco Dulbecco's modified Eagle's medium is a basal medium designed for the cultivation of a variety of mammalian cells. It provides a balanced salt solution and essential nutrients required for cell growth and maintenance.

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Gibco Dulbecco’s Modified Eagle Medium Gibco Dulbecco’s Modified Eagle Medium (DMEM), Gibco Dulbecco's Modified Eagle Medium: nutrient mixture F12 (DMEM/F12) Gibco Dulbecco's modified Eagle's medium (DMEM; Gibco Dulbecco modified Eagle medium Dulbecco’s modified

18 protocols using gibco dulbecco s modified eagle s medium

Hybrid Cell Line from Arabidopsis Cultures

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The human–Arabidopsis hybrid cell line was obtained from 60- and 149-day-old cultures by Wada et al. (2017). The 60-day-old and 149-day-old cell cultures originated from cell stocks frozen at −80 °C for 5.5 and 4.5 years, respectively. The 300-day-old culture originated from cells grown from the 149-day-old freeze-preserved culture. The hybrid cell line was cultured in Gibco Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with Gibco 10% fetal bovine serum (Thermo Fisher Scientific) and 6-μg/mL blasticidin S (KNF, Tokyo, Japan) at 37 °C in a 5% CO₂ incubator and subcultured every 2 to 3 days.

Liaw Y., Liu Y., Teo C., Cápal P., Wada N., Fukui K., Doležel J, & Ohmido N. (2021). Epigenetic Distribution of Recombinant Plant Chromosome Fragments in a Human–Arabidopsis Hybrid Cell Line. International Journal of Molecular Sciences, 22(11), 5426.

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Isolation and Culture of Primary Murine Hepatocytes

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The primary hepatocytes were obtained from the liver of anaesthetized male mice (6–7 weeks old) (Japan SLC Inc, Hamamatsu, Japan) by using a two-step hepatic portal vein perfusion method.21 (link),22 All procedures complied with the institutional ethical use protocol of the Animal Center in Tokyo Institute of Technology. After removal, the tissue was dissected in Hank’s solution and filtered through a 100 mm cell strainer. Then, the living hepatocytes were isolated and purified by using density gradient centrifugation. Subsequently, the cells were suspended in Gibco^® Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Waltham, MA, USA) with FBS (volume/volume [v/v] 0%, 1%, or 10%) and antibiotics (50 μg/mL penicillin, 50 μg/mL streptomycin, and 100 μg/mL neomycin). After the cellular viability was confirmed by using the Trypan Blue exclusion method (>95%), the viable cells were seeded on previously coated PVLA, gelatin, collagen, and coimmobilized surfaces. The primary hepatocytes were incubated at 37°C in an atmosphere of 5% CO₂ during culture. The medium was changed after 4 hours to remove the nonadherent and dead cells and was changed every day thereafter during culture.

Meng Q., Tao C., Qiu Z., Akaike T., Cui F, & Wang X. (2015). A hybrid substratum for primary hepatocyte culture that enhances hepatic functionality with low serum dependency. International Journal of Nanomedicine, 10, 2313-2323.

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Cell Line Culture and Western Blot Analysis

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The human GC cell lines, AGS, SGC-7901, MNK-45 and BGC-823, were purchased from China Infrastructure of Cell Line Resources (Beijing, China) and subsequently cultured in Gibco Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were cultured in a 5% CO_2, humidified incubator at 37°C. Goat polyclonal antibodies against Txr1 (1:100; cat. no., sc-244548), and mouse monoclonal antibodies against β-actin (1:1,000; cat. no., sc-8432) and TSP1 (1:100; cat. no., sc-59887) were acquired from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The mouse anti-ERCC1 monoclonal antibody (1:500; cat. no., MA5–13830) was purchased from Thermo Fisher Scientific, Inc.

LIU L., BAI Z., MA X., WANG T., YANG Y, & ZHANG Z. (2016). Effects of taxol resistance gene 1 expression on the chemosensitivity of SGC-7901 cells to oxaliplatin. Experimental and Therapeutic Medicine, 11(3), 846-852.

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Gefitinib and PI3K Inhibitor Protocol

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Gefitinib was purchased from AstraZeneca (Macclesfield, UK). The PI3K inhibitor, LY294002, was purchased from Sigma-Aldrich (St. Louis, MO, USA). Monoclonal human anti-mouse integrin β1 antibody (1:1,000; cat. no. CBL481P) was obtained from Chemicon® (Merck Millipore, Darmstadt, Germany). Monoclonal human anti-rabbit c-MET (1:1,000; cat. no. 8198), polyclonal human anti-rabbit Akt (1:1,000; cat. no. 9272), polyclonal human anti-rabbit phosphorylated (phospho)-Akt (Ser473; 1:1,000; cat. no. 9271), polyclonal human anti-rabbit Erk (1:1,000; cat. no. 4695), monoclonal human anti-rabbit phospho-Erk (1:1,000; cat. no. 4370), polyclonal human anti-rabbit EGFR (1:1,000; cat. no. 2232) and polyclonal human anti-rabbit phospho-EGFR (1:1,000; cat. no. 2234) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Monoclonal rabbit anti-mouse GAPDH antibody (1:10,000; cat. no. KC-5G5) was purchased from Shanghai Kangchen Bio-technology Co. (Shanghai, China). Gibco Dulbecco's modified Eagle's medium was from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

DENG Q.F., SU B., ZHAO Y.M., TANG L., ZHANG J, & ZHOU C.C. (2015). Integrin β1-mediated acquired gefitinib resistance in non-small cell lung cancer cells occurs via the phosphoinositide 3-kinase-dependent pathway. Oncology Letters, 11(1), 535-542.

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Neuroblastoma Cell Culture and Treatment

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Human neuroblastoma SH-SY5Y cells were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Gibco Dulbecco’s Modified Eagle’s medium (Catalog number: 11965092, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Thermo Fisher Scientific), 100 U/mL penicillin and 100 μg/mL streptomycin (Beyotime, Shanghai, China). The medium was subsequently changed every 2–3 days and cells were equilibrated in humidified air containing 5% CO₂ at 37°C. The cultured cells were treated with 100 μM 6-OHDA and/or 1 μM simvastatin at the same time, for different lengths of time, as described below.

Tong H., Zhang X., Meng X., Lu L., Mai D, & Qu S. (2018). Simvastatin Inhibits Activation of NADPH Oxidase/p38 MAPK Pathway and Enhances Expression of Antioxidant Protein in Parkinson Disease Models. Frontiers in Molecular Neuroscience, 11, 165.

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Recombinant EF-Tu Protein Expression

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The vectors pET30a-EF-Tu (from our own collection) and pGEX-6p-1 (TaKaRa, Dalian, China) were used to obtain recombinant His-tagged and glutathione S-transferase (GST)-tagged B. melitensis EF-Tu proteins, respectively. Escherichia coli Top10 cells (Invitrogen, Carlsbad, CA, United States) were used for all cloning experiments, while E. coli BL21(DE3) (Invitrogen) was used for the protein expression assay. Anti-EtMIC2 (microneme 2 protein from the protozoan parasite Eimeria tenella) McAbs were prepared in our laboratory as described previously (Liu et al., 2014 (link)). Mouse serum infected with B. melitensis artificially was kindly provided by Prof. Bu, Harbin Veterinary Research Institute. SP20 mouse myeloma cells were purchased from the American Type Culture Collection (Manassas, VA, United States) and cultured in Gibco Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, United States) supplemented with 10% (w/v) fetal bovine serum (FBS; Thermo Fisher Scientific). Female BALB/c mice aged 4–5 weeks were purchased from Vital River Laboratories (Beijing, China).

Zhao N., Jiang Y., Ming S., Liu S., Zhao X, & Wang F. (2019). Monoclonal Antibody Preparation and Epitope Identification for Brucella melitensis Elongation Factor Tu. Frontiers in Microbiology, 10, 1878.

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Culturing Human Melanoma Cell Lines

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The human melanoma A375-S2, SKMEL-28, SKMEL-5, MeWO and RPMI-7951 cell lines were purchased from the National Rodent Laboratory Animal Resource (Shanghai, China). All melanoma cell lines were grown in Gibco Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 100 units/ml Invitrogen penicillin-streptomycin (Thermo Fisher Scientific, Inc.). Normal human epidermal melanocytes (NHEMs) from adult skin (PromoCell GmbH, Heidelberg, Germany) were maintained in serum- and phorbol myristate acetate-free melanocyte growth medium M2 (PromoCell GmbH). The cell lines were cultured in a humidified incubator at 37°C in a 5% CO₂ and 95% air atmosphere for 2–3 days.

Qiu H., Yuan S, & Lu X. (2016). miR-186 suppressed CYLD expression and promoted cell proliferation in human melanoma. Oncology Letters, 12(4), 2301-2306.

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Establishing Tamoxifen-Resistant Breast Cancer Cell Lines

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The human breast cancer cell lines T47D and ZR75-1 were purchased from the American Type Culture Collection. They were cultured in Gibco Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific) with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin in a humidified incubator with 5% CO2 at 37°C.
TAM (4-hydroxytamoxifen, H7904) was purchased from Sigma-Aldrich and TAM-resistant cells (T47D-TAM^R and ZR75-1-TAM^R) were established by chronic exposure of the drug-sensitive T47D and ZR75-1 cells to stepwise increases in TAM concentrations, until a resistance concentration was achieved. The resistance to TAM in T47D-TAM^R and ZR75-1-TAM^R cells was confirmed by a Cell Counting Kit-8 (Dojindo Laboratories) which showed a higher viability of both cell types after TAM treatment compared with the sensitive cells (S1 Fig). The LD50 (50% lethal dose) in T47D and T47D-TAM^R cells was 4.0 μM and 6.3 μM, respectively, and in ZR75-1 and ZR75-1-TAM^R cells was 9.5 μM and 10.4 μM, respectively.

Hlaing M.T., Horimoto Y., Denda-Nagai K., Fujihira H., Noji M., Kaji H., Tomioka A., Ishizuka Y., Saeki H., Arakawa A., Saito M, & Irimura T. (2022). Tamoxifen-resistant breast cancer cells exhibit reactivity with Wisteria floribunda agglutinin. PLoS ONE, 17(8), e0273513.

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Transfection of HEK 293 Cells with Glycine Receptor Variants

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HEK 293 cells were obtained from the American Type Culture Collection and grown according to standard procedures (Freshney, 2002 (link)). Briefly, cells were cultured at 37°C and 5% CO₂ in Gibco^® Dulbecco’s modified Eagle’s medium with L-glutamine, sodium pyruvate, and 10% fetal bovine serum (Thermo-Fisher Scientific, Waltham, MA). Cells were split every 5 days with Gibco^® trypsin-EDTA (Thermo-Fisher Scientific) up to 25 times, after which new aliquots of early-passage cells were started. Cells were transfected with 4 µg of glycine receptor α1β, α2β, or α3β cDNA (1:20 α:β ratio) in modified pBK-cytomegalovirus vectors (Mihic et al., 1997 ) using PolyFect reagent (Qiagen, Valencia, CA). All cells were incubated for at least 48 h before use in panning.

Cornelison G.L., Pflanz N.C., Tipps M.E, & Mihic S.J. (2016). Identification and characterization of heptapeptide modulators of the glycine receptor. European journal of pharmacology, 780, 252-259.

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Vemurafenib Cytotoxicity in Melanoma Cells

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SK-mel 28 and SK-mel 24 cells were grown in Gibco Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, 11965–092), supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin and incubated at 37°C in 5% CO₂. Cell number was measured using a Cellometer Mini Cell Counter (Nexcelom Bioscience); the cell suspension was mixed at a 1:1 ratio with the trypan blue solution before the measurement. For all BRAFi experiments, SK-mel 28 and SK-mel 24 cells were treated with a 2 μM dose of vemurafenib, based on previous literature.^{22 (link)} For HP microcoil experiments, cells were plated 24 hours before treatment and incubated overnight to ensure cell adherence. SK-mel 28 and SK-mel 24 cells were treated with either vemurafenib or 0.1% dimethyl sulfoxide (DMSO, vehicle) for 24 or 48 hours. Immediately before the dissolution of HP [1-¹³C] pyruvate, cells were trypsinized, counted and divided into cell pellets of 2 × 10⁶ per sample. The order of samples loaded using the microcoil multi-shot protocol (Figure 1B) alternated between vemurafenib and vehicle treatment.

Lees H., Millan M., Ahamed F., Eskandari R., Granlund K.L., Jeong S, & Keshari K.R. (2020). Multi-sample measurement of hyperpolarized pyruvate-to-lactate flux in melanoma cells. NMR in biomedicine, 34(3), e4447.

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