Dmem f12

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DMEM/F12 is a cell culture medium developed by Thermo Fisher Scientific. It is a balanced salt solution that provides nutrients and growth factors essential for the cultivation of a variety of cell types, including adherent and suspension cells. The medium is formulated to support the proliferation and maintenance of cells in vitro.

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9 001 protocols using dmem f12

Optimizing Cell Culture Media Composition

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Eight types of culture media containing different concentrations and types of serum were used: (1) Dulbecco’s modified Eagle medium supplemented with F12 (DMEM/F12, Gibco, Carlsbad, CA, USA) and 5% fetal bovine serum (FBS) (Gibco); (2) DMEM/F12 with 10% FBS; (3) DMEM/F12 with 15% FBS; (4) DMEM/F12 with 20% FBS; (5) DMEM/F12 with 5% RS collected from adult male S–D rats; (6) DMEM/F12 with 10% RS; (7) DMEM/F12 with 15% RS; and (8) DMEM/F12 with 20% RS (Figure 10).

Lu M., Dong J., Lu T., Lv H., Yang P., Cheng Z., Li J., Liang B., Xu J., Li H, & He X. (2014). Effects of Different Sera Conditions on Olfactory Ensheathing Cells in Vitro. International Journal of Molecular Sciences, 16(1), 420-438.

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Co-culture of Breast Cancer and Stromal Cells

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MCF7 and CAF2 were seeded in 6 well plates at a ratio of 1:3 in DMEM/F-12 (ThermoFisher #11330032), 10% FBS (Corning #35–010-CV), 1% Penicillin and Streptomycin (Corning #30–001-CI) and DMEM (Corning #10-013) containing 10% FBS (Corning #35-010-CV) and 1% Penicillin, Streptomycin (Corning #30-001-CI), respectively. Treatment of MCF7 with estradiol (100 nM and 10 nM) was carried out after 24 h of starvation in DMEM/F-12 (ThermoFisher #11330032) containing 5% charcoal stripped serum (#12676029 Gibco) and 1% Penicillin and Streptomycin (Corning #30-001-CI). Transwells containing CAF2 were moved into MCF7 containing plates the following day. Proliferation rate was tested after 3 days of treatment.
To test the effect of JAK and TGFβ inhibition alone and in combination with 4-Hydroxytamoxifen, MCF7 were seeded in 6 well plates in DMEM/F-12 (ThermoFisher #11330032), 10% FBS (Corning #35-010-CV), 1% Penicillin and Streptomycin (Corning #30-001-CI). 4-Hydroxytamoxifen (Sigma-Aldrich #SML1666), SB-431542 (Tocris #1614) and Pyridone-6 (Tocris #6577) were added every other day for 5 days at final concentrations of 1 µM, 5 µM and 75 nM, respectively, Proliferation rate was tested after 5 days of treatment.
To test proliferation rate, the SRB assay (#S9012-56 Sigma) was performed following the protocol in [50 (link)]. Absorbance was quantified using the Synergy II microplate reader.

Reid S.E., Pantaleo J., Bolivar P., Bocci M., Sjölund J., Morsing M., Cordero E., Larsson S., Malmberg M., Seashore-Ludlow B, & Pietras K. (2024). Cancer-associated fibroblasts rewire the estrogen receptor response in luminal breast cancer, enabling estrogen independence. Oncogene, 43(15), 1113-1126.

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Primary Cortical Neuron Culture Protocol

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Primary neuronal cultures were performed as previously described [44 (link)]. Briefly, primary cortical neurons were obtained from a pregnant ICR mouse at E18 (Charles River Laboratories, Wilmington MA). Briefly, mice were euthanized, and embryos were retrieved. Cortical tissue was dissected from the mouse embryos, removed meninges around the brain. Cortical rinds were placed in PBS and cut into small pieces, 0.5–1 mm in length. After collection, cortical tissue was resuspended in 20 U/mL papain (Worthington Biochemical Corp., Lakewood NJ) and 1 mg/mL DNAse I (Worthington Biochemical Corp., Lakewood NJ) in DMEM/F12 (Thermo Fisher Scientific, Waltham MA). Tissue was digested at 37°C for 3–5 minutes, with occasional gentle trituration. Enzyme was neutralized in DMEM/F12 containing 0.5% v/v FBS (Thermo Fisher Scientific, Waltham MA) and cells were collected by centrifugation at 1000 rpm for 3 minutes. Cells were then seeded (density) and maintained for 3 days in DMEM/F12 containing N-2 and B-27 supplement (Thermo Fisher Scientific, Waltham MA) and 0.5% v/v FBS. All experimental procedures were approved by the Baylor College of Medicine Institutional Animal Care and use committee (IACUC) and performed according to the Animal Welfare Act and NIH guidelines for the care and use of animals.

Lopez-Silva T.L., Cristobal C.D., Edwin Lai C.S., Leyva-Aranda V., Lee H.K, & Hartgerink J.D. (2020). Self-assembling Multidomain Peptide Hydrogels accelerate Peripheral Nerve Regeneration after Crush Injury. Biomaterials, 265, 120401.

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Isolation of Stromal Vascular Fraction

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SVF was isolated from subcutaneous adipose tissue of 3 obese patients, who underwent plastic surgery (abdominoplasty). Briefly, freshly collected subcutaneous adipose tissue was separated from major vessels and fibers, minced and digested with 1 mg/ml collagenase type II (Sigma-Aldrich) in DMEM-F12 (ThermoFisher) at 37 °C for 1 h. Cell suspension containing SVF was centrifuged (350×g, 8 min), pellet was resuspended in erythrocyte-lysing buffer for 5 min, washed in DMEM-F12 3% FBS, filtered with a 100 μm cell strainer, centrifuged (350×g, 8 min) and resuspended in DMEM-F12 10% FBS for further analysis.

Coletta S., Salvi V., Della Bella C., Bertocco A., Lonardi S., Trevellin E., Fassan M., D’Elios M.M., Vermi W., Vettor R., Cagnin S., Sozzani S., Codolo G, & de Bernard M. (2020). The immune receptor CD300e negatively regulates T cell activation by impairing the STAT1-dependent antigen presentation. Scientific Reports, 10, 16501.

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Organoid Culture Conditions Comparison

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Explant medium (ExM): DMEM/F12 (Thermo, 11330032) containing B27 supplement (no vitamin A; Invitrogen, Paisley, UK, 12587010), 2 mM L-glutamine (SIGMA, G7513) and 100 U/mL Penicillin/100 µg/mL Streptomycin (SIGMA, P0781).
Clevers’ medium (CM[28 (link)]): DMEM/F12 containing 5% R-spondin conditioned medium, 5 nM neuregulin (Peprotech, 100–03), 5 ng/mL epidermal growth factor (Peprotech, AF-100–15), 100 ng/mL noggin (Peprotech, 120-10C), 500 nM A83-01 (Tocris, 2939), 5 µM Y27632 (Abmole, S1049), 500 nM SB202190 (Sigma, S7067), 1 × B27 (with vitamin A, Gibco, 1750444), 1.25 nM N-acetylcysteine (Sigma, A9165), 5 mM nicotinamide (Sigma, N0636), 1 × Glutamax (Invitrogen, 12634–034), 10 mM HEPES (Invitrogen, 15,630–056), 100 U/mL Penicillin/100 µg/mL Streptomycin and 50 ng/mL FGF2 (Thermo, 100-18B).
FCS medium: DMEM/F12 and 10% foetal calf serum (FCS, Thermo, 10270106), 100 U/mL Penicillin/100 µg/mL Streptomycin.

Wilby A.J., Cabral S., Zoghi N., Howell S.J., Farnie G, & Harrison H. (2024). A novel preclinical model of the normal human breast. Journal of Mammary Gland Biology and Neoplasia, 29(1), 9.

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Neonatal Rat Hippocampal Neuron Culture

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The hippocampus was dissected from neonatal rats (7 days old), triturated, and dissociated through trypsin. The dissociated cells were filtered and centrifuged and then resuspended in Dulbecco's Modified Eagle Medium/F12 medium (DMEM/F12, Thermo‐Scientific, MA, USA). Then, the cells were seeded onto dishes coated with poly‐D‐lysine and cultured with DMEM/F12 supplemented with 10% fetal bovine serum (FBS, Thermo Scientific, MA, USA), 1% glutamine, 4.5 g/L B27 plus glucose, and 1% penicillin–streptomycin (Sigma–Aldrich, MI, USA). After culturing for 3 days, 5 μg/ml cytosine arabinoside C (Sigma–Aldrich, MI, USA) was added to the medium and cultured for 24 h. The neurons were cultured in a humidified incubator at 37°C and 5% CO₂ for 14 days.

Chen Y., Gao X, & Pei H. (2022). miRNA‐384‐3p alleviates sevoflurane‐induced nerve injury by inhibiting Aak1 kinase in neonatal rats. Brain and Behavior, 12(7), e2556.

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Embryoid Body Formation from hiPSCs

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Embryoid bodies (EBs) were generated from hiPSCs by adapting the methods reported in a previous study⁵⁵ (link) with modifications. Briefly, hiPSCs were seeded on AggreWell 800 Plates (STEMCELL) with UM/F- medium supplied with 10 μM Y-27632 (Enzo Life Sciences). UM/F- medium composed of DMEM/F12 (Thermo Fisher Scientific) containing 20% Knockout Serum Replacement (Thermo Fisher Scientific), 1× MEM nonessential amino acids (Thermo Fisher Scientific), 1x GlutaMAX (Thermo Fisher Scientific) and 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific). hiPSCs were cultured with UM/F- medium supplied with 10 μM Y-27632 for 5 days (medium refreshed every two days) to allow the formation of spheroids. On day 6, EBs were resuspended in fresh UM/F- medium supplied with 10 μM Y-27632 and transferred to regular cell culture plates. On the next day (differentiation day 1), EBs were cultured with EB20 medium composed of DMEM/F12 containing 20% FBS (Thermo Fisher Scientific), 1× MEM nonessential amino acids, 1x GlutaMAX and 0.1 mM β-mercaptoethanol. For CHIR99021 treatment, 6 μM CHIR99021 was supplemented to EB20 medium from differentiation day 2 to day 4.

Chi C., Knight W.E., Riching A.S., Zhang Z., Tatavosian R., Zhuang Y., Moldovan R., Rachubinski A.L., Gao D., Xu H., Espinosa J.M, & Song K. (2023). Interferon hyperactivity impairs cardiogenesis in Down syndrome via downregulation of canonical Wnt signaling. iScience, 26(7), 107012.

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Oleic Acid-Induced Liver Cell Model

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HepG2 was maintained in DMEM/F12 (Thermo Fisher Scientific Inc.) containing 10% FBS. THP-1 cell was maintained in RPMI1640 (Thermo Fisher Scientific Inc.) or DMEM/F12 containing 10% FBS. LX-2 was maintained in DMEM High Glucose (Thermo Fisher Scientific Inc.) containing 2% FBS and 1× Glutamine. 100,000 cells were seeded into 96 well round bottom microwell plate, centrifuged, and cultured overnight. For mix condition, HepG2, THP-1, and LX-2 were seeded at a 1:1:1 ratio (total cell number is 100,000). After 1 day culture, 800 μM OA was added to the media. The cells were cultured for 3 days (for lipids accumulation and inflammation test) and 5 days (for fibrosis test), and assayed for further analysis.

Ouchi R., Togo S., Kimura M., Shinozawa T., Koido M., Koike H., Thompson W., Karns R., Mayhew C., McGrath P.S., McCauley H.A., Zhang R.R., Lewis K., Hakozaki S., Ferguson A., Saiki N., Yoneyama Y., Takeuchi I., Mabuchi Y., Akazawa C., Yoshikawa H.Y., Wells J.M, & Takebe T. (2019). Modeling Steatohepatitis in Humans with Pluripotent Stem Cell-Derived Organoids. Cell metabolism, 30(2), 374-384.e6.

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Subculturing Neural Stem Cells

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At 7 days post-culture, the diameter of neurospheres was commonly ~100 µm and subculturing was performed. In detail, NSCs were digested using 0.25% trypsin (1:250, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C for 10 min and DMEM/F12 (1:1; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing serum was used to stop the digestion. NSC suspension was collected into a 15 ml centrifuge tube. Subsequently, centrifugation at 560 × g (4°C) was performed for 5 min. The supernatant was discarded. The cell suspension was resuspended in DMEM/F12 (1:1; Gibco; Thermo Fisher Scientific, Inc.) containing 2% B-27 (Gibco; Thermo Fisher Scientific, Inc.), 20 ng/ml bFGF (R&D Systems, Inc.), 20 ng/ml EGF (R&D Systems, Inc.), 2 mmol/l glutamine (Gibco; Thermo Fisher Scientific, Inc.), 10,000 U/l penicillin and 10 mg/l streptomycin. The cellular density was adjusted to 1.5–2.5×10⁶/ml and inoculated into a culture bottle (25 ml in volume), which was gently swayed for even distribution. The cells were incubated in an incubator at 37°C.

Hu Y.D., Zhao Q., Zhang X.R., Xiong L.L., Zhang Z.B., Zhang P., Zhang R.P, & Wang T.H. (2018). Comparison of the properties of neural stem cells of the hippocampus in the tree shrew and rat in vitro. Molecular Medicine Reports, 17(4), 5676-5683.

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Cell Culture Conditions for Cancer and Fibroblast Lines

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Human breast cancer cell line MCF7 and human lung adenocarcinoma cell line A549 were kindly provided by Dr. S. Dmitriev, immortalized human fibroblasts cell line VA13 was kindly provided by Dr. M. Rubtsova, human embryonic kidney HEK293T cell line was kindly provided by Dr. E. Knyazhanskaya. MCF7, VA13, A549, and HEK293T cell lines were maintained in DMEM/F-12 (Thermo Fisher Scientific, Waltham, MA, USA) culture medium containing 10% fetal bovine serum (Thermo Fisher Scientific, USA) and 50 µg/mL penicillin and 0.05 mg/mL streptomycin at 37 °C (Thermo Fisher Scientific, USA) in 5% CO2. Medium F-12 (Paneco LLC, Russia) containing 10% fetal bovine serum (Thermo Fisher Scientific, USA), 50 µg/mL penicillin, and 0.05 mg/mL streptomycin (Thermo Fisher Scientific, USA) was used instead of DMEM/F-12 in some cytotoxicity assays. PC-3 cell line (ATCC) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine (Gibco, New York, NY, USA). Cells were maintained at 37 °C in a humidified incubator MCO-18AC (Sanyo, Osaka, Japan) supplied with 5% CO₂. After attaining 80% confluence, the cells were harvested with TrypLE (Gibco) and subcultured 1:8. Cell cultures were tested for the absence of mycoplasma.

Filatov V.E., Iuzabchuk D.A., Tafeenko V.A., Grishin Y.K., Roznyatovsky V.A., Lukianov D.A., Fedotova Y.A., Sukonnikov M.A., Skvortsov D.A., Zyk N.V, & Beloglazkina E.K. (2022). Dispirooxindole-β-Lactams: Synthesis via Staudinger Ketene-Imine Cycloaddition and Biological Evaluation. International Journal of Molecular Sciences, 23(12), 6666.

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