Dmem f12 1 1 media

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DMEM/F12 (1:1) media is a commonly used cell culture medium that provides nutrients and support for the growth and maintenance of a variety of cell types. It is a 1:1 mixture of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture, which together provide a balanced combination of amino acids, vitamins, salts, and other essential components required for cell culture.

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DMEM/F12 (9007 citations) DMEM media (655 citations) DMEM/F12 media (463 citations) DMEM/F12 1:1 (101 citations) F12 media (67 citations) DMEM/F12 (1:1) cell culture media (4 citations) Serum-free (SF) media (3:1 DMEM/F12 mixture) (2 citations)

27 protocols using dmem f12 1 1 media

Xenograft-Derived Tumor Sphere Assay

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All animal experiments were approved by the Johns Hopkins University Animal Care and Use Committee. Low passage xenografts were generated from primary surgical specimens collected at the Johns Hopkins Hospital, serially passaged in nude mice, and prepared as single cell suspensions as previously described (5 (link), 27 (link)). Tumor spheres enriched in CSCs were generated by culturing cells (200,000 cells/ml) in 60-mm ultralow attachment plates (Corning) and DMEM/F-12 (1/1) media (Invitrogen) supplemented with 1X B-27 supplement (Thermo Fisher Scientific), 20ng/ml bFGF (Invitrogen), 1% penicillin-streptomycin and 1% L-glutamine. Tumor cell differentiation was induced by culturing cells in complete media containing 10% FBS.
Tumor-initiating cell (TIC) frequency was determined by injecting L3.6pl cells (100,000) in serum-free DMEM and Matrigel (BD Biosciences) subcutaneously into the flanks of NSG mice. Following tumor formation, mice were provided doxycycline (2mg/ml, Sigma-Aldrich) in 5% sucrose. Following 9 days of treatment, tumors were collected, dissociated, and depleted of mouse cells then injected (50, 100, 250, and 500 cells) into secondary recipients. Tumor formation was monitored for 30 days, and (TIC) frequency was calculated using extreme limiting dilution analysis (ELDA) (28 (link)).

Penchev V.R., Chang Y.T., Begum A., Ewachiw T., Gocke C., Li J., McMillan R.H., Wang Q., Anders R., Marchionni L., Maitra A., Uren A., Rasheed Z, & Matsui W. (2019). Ezrin promotes stem cell properties in pancreatic ductal adenocarcinoma. Molecular cancer research : MCR, 17(4), 929-936.

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Embryonic Cerebral Cortex Slice Culture

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Slice culture of embryonic cerebral cortices was performed as described previously (Nishimura et al, 2010 (link)). E16 embryonic brains, electroporated at E14, were cut into 300 μm coronal slices with a microtome (Leica) in DMEM/F‐12 1:1 media (Invitrogen). Cortical slices were cultured on the insert membranes (Millipore) in 2 ml of enriched media (100 μg/ml transferrin, 25 μg/ml insulin, 20 nM progesterone, 60 μM putrescine, 10 ng/ml EGF, 10 ng/ml bFGF, 5% Fetal bovine serum and 5% Horse serum; Miyata et al, 2002 (link)) in a CO₂/O₂ incubator (37°C, 5% CO₂, 40 or 60% O₂) under confocal laser scanning time‐lapse microscopy, FV1000 (Olympus) or TCL‐SP2 (Leica).

Shikanai M., Ito S., Nishimura Y.V., Akagawa R., Fukuda M., Yuzaki M., Nabeshima Y, & Kawauchi T. (2023). Rab21 regulates caveolin‐1‐mediated endocytic trafficking to promote immature neurite pruning. EMBO Reports, 24(3), e54701.

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Primary Murine Kidney Cell Isolation

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For primary kidney cell preparation, kidneys from 8- to 9-month-old mice were collected and washed in cold HBSS (Hank Balances Salt Solution, Gibco) to remove residual blood. Renal fibrous capsule, connective tissue and renal medulla were removed. Kidney cortical tissue was dissected and mechanically minced using scalpels. Tissue homogenates were then transferred into 50 ml falcon tubes containing HBSS with 1 mg/ml Collagenase IV (Invitrogen) to allow digestion at 37°C for 30 min. Digested kidney fragments were then passed through a 100 μm sieve in another falcon tube to remove fibrous tissue. The sieve was washed in HBSS and the cell suspension centrifuged at 1200 rpm for 5 min two times. Finally, the cell pellet was resuspended in complete culture media: DMEM/F12 1:1 media (Invitrogen) containing 5% FBS, 10 ng/ml epidermal growth factor (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 1% l-glutamine (Invitrogen), 50 mM hydrocortisone (Sigma Aldrich), 5 μg/ml insulin (Invitrogen), 5 μg/ml transferrin (Sigma Aldrich) and 50 nM sodium selenite (Sigma Aldrich). Cells were cultured in 75 cm² plastic flasks with complete culture medium at 37°C under 5% CO₂ in a humidified atmosphere.

Pellegrini L., Hauser D.N., Li Y., Mamais A., Beilina A., Kumaran R., Wetzel A., Nixon-Abell J., Heaton G., Rudenko I., Alkaslasi M., Ivanina N., Melrose H.L., Cookson M.R, & Harvey K. (2018). Proteomic analysis reveals co-ordinated alterations in protein synthesis and degradation pathways in LRRK2 knockout mice. Human Molecular Genetics, 27(18), 3257-3271.

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Mesenchymal Precursor Isolation and Adipogenesis

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Mesenchymal precursors were isolated from ears of mice of the indicated genotypes, as previously described [53] (link). Cells were maintained in 5% CO₂ and DMEM/F12 1:1 media (Gibco; Invitrogen) supplemented with 10% FBS (Cytiva HyClone), primocin (InVivoGen), and 10 ng/mL recombinant bFGF (PeproTech). For induction of adipogenesis, recombinant bFGF was removed and replaced with 10% FBS containing 0.5 mM methylisobutylxanthine, 1 μM dexamethasone, 5 μg/mL insulin, and 5 μM rosiglitazone. On day 2, cells were fed 5 μg/mL insulin plus 5 μM troglitazone. On day 4 and every 2 days thereafter, cells were fed with 10% FBS-supplemented media. All the media conditions used in the experiments within this study contain 10% FBS, unless otherwise specified in the individual figure legends. When using 2% FBS, Insulin-Transferrin-Selenium (ITS) (Gibco; Invitrogen) was supplemented.

Mori H., Peterson S.K., Simmermon R.C., Overmyer K.A., Nishii A., Paulsson E., Li Z., Jen A., Uranga R.M., Maung J.N., Yacawych W.T., Lewis K.T., Schill R.L., Hetrick T., Seino R., Inoki K., Coon J.J, & MacDougald O.A. (2024). Scd1 and monounsaturated lipids are required for autophagy and survival of adipocytes. Molecular Metabolism, 83, 101916.

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SH-SY5Y Neuroblastoma Cell Culture

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A neuroblastoma cell line SH-SY5Y (ATCC) was grown in DMEM/F12 (1: 1) media (Invitrogen) routinely supplemented with 10% FBS, 100 units/ml of penicillin, and 100 μg/ml of streptomycin. The culture was incubated at 37°C under an atmosphere of 5% CO₂. Prior to treatment, cells were removed from the medium and incubated serum-free for 24 hours.

Ko S.Y., Ko H.A., Chu K.H., Shieh T.M., Chi T.C., Chen H.I., Chang W.C, & Chang S.S. (2015). The Possible Mechanism of Advanced Glycation End Products (AGEs) for Alzheimer’s Disease. PLoS ONE, 10(11), e0143345.

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Cell Culture and Pharmacological Inhibition

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5637, BIU87, T24 and EJ cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured at 37 °C in 5% CO₂ in DMEM-F12 (1:1) media (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 1% penicillin (100 U/ml, Invitrogen Life Technologies), 1% streptomycin (100 μg/ml, Invitrogen Life Technologies), L-glutamine (292 μg/ml, Invitrogen Life Technologies) and 10% fetal bovine serum (HyClone Laboratories, Logan, UT, USA). GSK690693 (Sigma-Aldrich, St. Louis, MO, USA; 5 μM) or MK-2206 (Selleckchem, Houston, TX, USA; 5 μM) was added to the media at the indicated times. For transient gene transfection of cells, Lipofectamine 2000 (Invitrogen Life Technologies) was used.

Jiang L., Dong P., Zhang Z., Li C., Li Y., Liao Y., Li X., Wu Z., Guo S., Mai S., Xie D., Liu Z, & Zhou F. (2015). Akt phosphorylates Prohibitin 1 to mediate its mitochondrial localization and promote proliferation of bladder cancer cells. Cell Death & Disease, 6(2), e1660-.

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Temperature-Induced Adipocyte Differentiation

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Mesenchymal precursors were isolated from ears of mice of the indicated genotypes, as previously described [18 (link)]. Cells were maintained in 5% CO₂ and DMEM/F12 1:1 media (Gibco; Invitrogen, Carlsbad, CA, USA) supplemented with 15% FBS (Atlas Biologicals, Fort Collins, CO, USA), primocin (InVivoGen, San Diego, CA, USA), and 10 ng/ml recombinant bFGF (PeproTech, Rocky Hill, NJ, USA). For induction of adipogenesis, recombinant bFGF was removed and replaced with 10% FBS containing 0.5 mM methylisobutylxanthine, 1 μM dexamethasone, 5 μg/ml insulin, and 5 μM troglitazone. On day 2, cells were fed 5 μg/ml insulin plus 5 μM troglitazone. On day 4 and every 2 days thereafter, cells were fed with 10% FBS. At day 4 of differentiation, plates of adipocytes were either kept at 37°C or moved to another incubator maintained at 31°C. Of note, since adipocyte culture media contains both sodium bicarbonate and HEFPES, pH changes are unlikely to contribute to temperature-induced effects on adipocyte gene expression.

Mori H., Dugan C.E., Nishii A., Benchamana A., Li Z., Cadenhead TS I.V., Das A.K., Evans C.R., Overmyer K.A., Romanelli S.M., Peterson S.K., Bagchi D.P., Corsa C.A., Hardij J., Learman B.S., El Azzouny M., Coon J.J., Inoki K, & MacDougald O.A. (2021). The molecular and metabolic program by which white adipocytes adapt to cool physiologic temperatures. PLoS Biology, 19(5), e3000988.

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Cell Culture Protocols for Cancer Cell Lines

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IGROV1 cells were cultured in RPMI1640 medium (Biosource, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Biosource), 50 units/mL penicillin, and 50 μg/mL streptomycin. MDAMB468 cells were grown in DMEM/F12 (1:1) media (Biosource) supplemented with 10% FBS (Biosource), 50 units/mL penicillin and 50 μg/mL streptomycin. LNCaP cells were cultured in either RPMI1640 medium (Biosource) supplemented with 10% FBS (Biosource), 50 units/mL penicillin, and 50 μg/mL streptomycin, or in androgen-deprived media i.e. phenol red-free RPMI1640 medium (Biosource) supplemented with 10% charcoal stripped FBS (CSS, Gemini, West Sacramento, CA), 50 units/mL penicillin and 50 μg/mL streptomycin. Cultures treated with EGF were starved for at least 3 hours in serum-free medium (SFM) because this eliminates the background effect of endogenous EGF that may be present in the serum, in order to optimize the effects of endogenously added EGF.

Xie Y., Nakanishi T., Natarajan K., Safren L., Hamburger A.W., Hussain A, & Ross D.D. (2015). Functional cyclic AMP response element in the breast cancer resistance protein (BCRP/ABCG2) promoter modulates epidermal growth factor receptor pathway- or androgen withdrawal-mediated BCRP/ABCG2 transcription in human cancer cells. Biochimica et biophysica acta, 1849(3), 317-327.

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Cell Culture Conditions for Prostate Cancer

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VCaP cells (obtained from ATCC, used at passage numbers less than 40) were grown in DMEM/F12 (1:1) media (BioSource, Grand Island, NY, USA) supplemented with 5% FBS (Biosource), 50 units/mL penicillin, and 50 µg/mL streptomycin. LNCaP cells (obtained from ATCC, used at passage numbers less than 30) were cultured in RPMI1640 medium (BioSource, Grand Island, NY, USA) supplemented with 10% FBS (Biosource), 50 units/mL penicillin, and 50 µg/mL streptomycin. The culture medium was changed every other day. For all cell culture experiments, the cell passage number of wild-type LNCaP cells was 34 or less while that for VCaP cells was 50 or less.

Xie Y., Wang L., Khan M.A., Hamburger A.W., Guang W., Passaniti A., Munir K., Ross D.D., Dean M, & Hussain A. (2021). Metformin and Androgen Receptor-Axis-Targeted (ARAT) Agents Induce Two PARP-1-Dependent Cell Death Pathways in Androgen-Sensitive Human Prostate Cancer Cells. Cancers, 13(4), 633.

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Culturing Immortalized Human Retinal Pigment Epithelial Cells

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hTERT-RPE-1 cells were obtained from ATCC (VA, USA, CRL-4000) and were cultured under standard mammalian cell conditions with 10% FBS (Gibco, ThermoFisher Scientific) and 1% antibiotics in DMEM/F12 (1:1) media (Gibco). hTERT-RPE-1 cells were authenticated by STR profiling service from Cosmogenetech Inc (Seoul, Republic of Korea). Mycoplasma contamination was monitored with Cell Culture Contamination Detection Kit (Invitrogen, ThermoFisher Scientific) regularly.

Jang D.G., Kwon K.Y., Kweon Y.C., Kim B.G., Myung K., Lee H.S., Young Park C., Kwon T, & Park T.J. (2022). GJA1 depletion causes ciliary defects by affecting Rab11 trafficking to the ciliary base. eLife, 11, e81016.

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