Dulbecco s modified eagle medium f12 medium

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Dulbecco's Modified Eagle Medium/F12 medium is a widely used cell culture medium formulation that supports the growth and maintenance of a variety of mammalian cell types. It is a balanced salt solution that provides essential nutrients, vitamins, and amino acids required for cell proliferation and survival.

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13 protocols using dulbecco s modified eagle medium f12 medium

Cellular Uptake of T3 in COS-1 and JEG-3 Cells

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COS-1 African green monkey kidney (CVCL_0223) and JEG-3 human choriocarcinoma (CVCL_0363) cells were obtained from ECACC (Sigma-Aldrich) and were cultured and transiently transfected as previously described (20 (link)). For T3 uptake studies, COS-1 or JEG-3 cells were cultured in 24-well plates, and transiently transfected at 70% confluence with 100 ng pcDNA3 empty vector (EV), or 100 ng WT or indicated mutant MCT8 expression construct in the presence or absence of 50 ng CRYM. Kinetic studies were performed in the absence of CRYM. For surface biotinylation studies, cells were seeded in 6-well plates (6 wells per condition, which were pooled during lysate preparation) and transiently transfected with 500 ng pcDNA3 EV, WT, or indicated mutant MCT8 per well. For immunocytochemistry, JEG-3 cells were cultured in 24-well dishes on 10-mm glass coverslips coated with poly-D-lysine (Sigma-Aldrich).
Human fibroblasts were derived from skin biopsies and cultured in Dulbecco’s modified Eagle medium/F12 medium (Invitrogen, Breda, NL), containing 9% heat-inactivated fetal bovine serum (Invitrogen), 2% penicillin/streptomycin (Roche, Woerden, The Netherlands). For uptake studies, fibroblasts were seeded in 6-well plates and grown until >95% confluence (27 (link)).

van Geest F.S., Meima M.E., Stuurman K.E., Wolf N.I., van der Knaap M.S., Lorea C.F., Poswar F.O., Vairo F., Brunetti-Pierri N., Cappuccio G., Bakhtiani P., de Munnik S.A., Peeters R.P., Visser W.E, & Groeneweg S. (2020). Clinical and Functional Consequences of C-Terminal Variants in MCT8: A Case Series. The Journal of Clinical Endocrinology and Metabolism, 106(2), 539-553.

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Isolation and Dissociation of Rat Dorsal Root Ganglia

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Experiments were carried out in accordance with the National Institutes of Health
Guidelines for the Care and Use of Laboratory Animals; all animal protocols were
approved by the Institutional Animal Care and Use Committee of the Veterans
Affairs Connecticut Health care System, West Haven, Connecticut. Adult
Sprague–Dawley rats (four- to six-week old) were deeply anesthetized by
CO₂ narcosis and decapitated. DRGs were isolated and dissociated
after anesthesia. In brief, dissected ganglia were placed in ice-cold oxygenated
complete saline solution containing the following (in mM): 137 NaCl, 5.3 KCl, 1
MgCl₂, 25 sorbitol, 3 CaCl₂, and 10 HEPES, pH 7.2.
DRGs were digested for 20 min at 37°C in complete saline solution containing
collagenase D (1.5 mg/ml) and papain (30 U/ml). DRGs were centrifuged and
resuspended in DRG media (Dulbecco’s Modified Eagle Medium/F12 medium containing
100 U/ml penicillin, 0.1 mg/ml streptomycin (Invitrogen, Carlsbad, CA), 10%
fetal bovine serum (Sigma-Aldrich, St. Louis, MO), 1.5 mg/ml bovine serum
albumin and 1.5 mg/ml trypsin inhibitor (Sigma)). DRGs were triturated in DRG
media and centrifuged. The cell pellet was resuspended in DRG media, placed on
top of a layer of 15% bovine serum albumin in DRG media and centrifuged at 200
relative centrifugal force for 10 min to remove non-neuronal cells.

Huang Y., Wen L.L., Xie J.D., Ouyang H.D., Chen D.T, & Zeng W.A. (2019). Antinociceptive effectiveness of the inhibition of NCX reverse-mode action in rodent neuropathic pain model. Molecular Pain, 15, 1744806919864511.

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Functional Knockdown of Key Regulators in CML and 293T Cells

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Human CML K562 cells and 293T cells were cultured in Dulbecco’s modified Eagle medium/F12 medium (Invitrogen, Carlsbad, CA) containing 10% newborn bovine serum at 37°C and 5% CO₂ in an incubator and passaged every 2–3 days. Cells in the logarithmic growth phase and with a trypan blue rejection rate > 95% were used in all experiments. The cells were transfected with small interfering RNA (siRNA) and miRNA mimic sequences according to the instructions of the Lipofectamine 2000 Transfection Kit (Thermo Fisher Scientific, Waltham, MA). The following experimental groups were used: cell blank control group (cells); negative control (NC) group (NC sequence + Lipofectamine 2000); si-NC group (siRNA for NC sequence + Lipofectamine 2000); si-EGR1 group (siRNA sequence for EGR1 + Lipofectamine 2000); miR-203a mimic group (miR-203a mimic + Lipofectamine 2000); si-WT1 group (siRNA sequence for WT1 + Lipofectamine 2000); si-BMI1 group (siRNA sequence for BMI1 + Lipofectamine 2000); and si-XIAP group (siRNA sequence for XIAP + Lipofectamine 2000). The siRNA sequences are shown in Table 1. The sequence for miR-203a was obtained from the website of the National Center for Biotechnology Information³.

He J., Han Z., An Z., Li Y., Xie X., Zhou J., He S., Lv Y., He M., Qu H., Liu G, & Li Y. (2021). The miR-203a Regulatory Network Affects the Proliferation of Chronic Myeloid Leukemia K562 Cells. Frontiers in Cell and Developmental Biology, 9, 616711.

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HCC Tumoursphere Formation Assay

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After completing the designated intervention, HCC cells were plated at a density of 5000 cells per well in six-well ultra-low attachment plates (Corning, Corning, NY, USA) and then cultured with serum-free Dulbecco’s Modified Eagle Medium/F12 medium (Gibco) supplemented with 20 ng/mL human EGF, 1% B27 (Invitrogen, Carlsbad, CA, USA) and 20 ng/mL fibroblast growth factor. Subsequently, HCC cells were incubated at 37 °C with 5% CO₂ for 14 days. A microscope (Nikon Instruments Inc.) was used to count the number and determine the diameter of the tumourspheres at a magnification of ×200.

Sun L., Wang L., Chen T., Shi Y., Yao B., Liu Z., Wang Y., Li Q., Liu R., Niu Y., Tu K, & Liu Q. (2020). LncRNA RUNX1-IT1 which is downregulated by hypoxia-driven histone deacetylase 3 represses proliferation and cancer stem-like properties in hepatocellular carcinoma cells. Cell Death & Disease, 11(2), 95.

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Isolation and Culture of Neural Stem Cells

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Adult male C57BL/7 mice (8–9-week old) were obtained from ORIENT BIO (Seongnam, Korea). All experiments were approved by and carried out in accordance with the regulations of the Animal Care and Use Committee of Korea University. The SVZ or SCZ area was isolated from each adult mouse brain and digested with 0.8% papain (Worthington, Lakewood, NJ, USA) and 0.08% dispase II (Roche Applied Science, Indianapolis, IN, USA) in Hank’s Balanced Salt Solution for 45 min at 37°C as described previously (Kim et al. 2016 ). Digested cells were seeded in an ultra-low attachment surface dish and maintained in suspension culture with Dulbecco's Modified Eagle Medium/F12 medium containing 1% N2, 2% B27 supplement (Gibco BRL, Franklin Lakes, NJ, USA), and penicillin-streptomycin. Growth factors including basic fibroblast growth factor (bFGF, 20 ng/ml; Invitrogen, Carlsbad, CA, USA), epidermal growth factor (EGF, 20 ng/ml; Invitrogen), and l-ascorbic acid (20 ng/ml; Sigma-Aldrich, St. Louis, MO, USA) were added to each culture every day. Generated neurospheres were passaged by dissociating the neurospheres into single cells with Accutase (Innovative Cell Technologies, San Diego, CA, USA) for 10 min at 37°C.

Kim J.Y., Shaker M.R., Lee J.H., Lee B., Kim H, & Sun W. (2017). Identification of molecular markers distinguishing adult neural stem cells in the subventricular and subcallosal zones. Animal Cells and Systems, 21(3), 152-159.

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Generating Isogenic Cell Lines for XPV

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The XPPHBE (Coriell Cell Repository no. GM02449) Epstein–Barr Virus-transformed XPV lymphoblast cell line (XPV-L Pol eta^−/−) was grown in Roswell Park Memorial Institute medium supplemented with 15% fetal bovine serum (FBS) and 1% penicillin and streptomycin. The XP30RO SV40-transformed XPV fibroblast cell line (XPV-F Pol eta^−/−) was grown in Dulbecco’s Modified Eagle Medium/F12 medium (Gibco) supplemented with 15% FBS and 1% penicillin and streptomycin. XPV fibroblasts stably complemented with WT Pol eta (XPV-F + Pol eta) were generated by complementing the XPV-F Pol eta^−/− cell line with the Pol eta cDNA [pcDNA 3.1 zeo(−)] and selected in 100 µg/mL Zeocin (30 (link)). No detectable Pol eta protein expression was observed in the XPV-F Pol eta^−/− cell line, but a significantly high level of Pol eta protein expression was detected in the XPV-F + Pol eta cell line (30 (link)). The XPV-F Pol eta^−/− and XPV-F + Pol eta cell lines were provided by Kristin A. Eckert, Pennsylvania State University College of Medicine, Hershey, PA.

Twayana S., Bacolla A., Barreto-Galvez A., De-Paula R.B., Drosopoulos W.C., Kosiyatrakul S.T., Bouhassira E.E., Tainer J.A., Madireddy A, & Schildkraut C.L. (2021). Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites. Proceedings of the National Academy of Sciences of the United States of America, 118(48), e2106477118.

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Evaluating Eupatilin's Anti-Fibrotic Effects on Ishikawa Cells

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Ishikawa cells (a well-differentiated human endometrial adenocarcinoma cell line), obtained from American Type Cell Culture (Manassas, VA, USA), were maintained in Dulbecco’s Modified Eagle Medium/F12 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco), 100 mg/mL streptomycin (Gibco) and 2 mM L-glutamine (Gibco). Recombinant human TGF-β (cat# L1718; Peprotech, Rocky Hill, NJ, USA) was used at concentrations of 1, 5, or 10 ng/mL. The anti-fibrotic effects of eupatilin (cat# S3846; Selleckchem, Houston, TX, USA) were examined at final concentrations of 25, 50, and 100 μM. Cells were grown on a Matrigel-coated cover glass (1:8 dilution, growth factor-reduced; Corning, Tewksbury, MA, USA) for further investigation of morphological changes and immunofluorescence (IF) analyses.

Lee C.J., Hong S.H., Yoon M.J., Lee K.A., Choi D.H., Kwon H., Ko J.J., Koo H.S, & Kang Y.J. (2020). Eupatilin treatment inhibits transforming growth factor beta-induced endometrial fibrosis in vitro. Clinical and Experimental Reproductive Medicine, 47(2), 108-113.

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In vitro NAFLD and Intermittent Hypoxia

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The hepatocytes were cultured in Dulbecco’s Modified Eagle Medium/F12 medium (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) in a 5% CO₂ incubator at 37 °C.
To induce NAFLD in vitro, a free fatty acid (FFA) mixture (oleate: palmitate = 2:1) (0.5 mmol/L concentration) was added to the culture medium and incubated for 24 h. Additionally, 20 µg/mL of exosomes were added to the medium to induce the hepatocytes.
To induce intermitted hypoxia in vitro [26 ], the O₂ concentration in the incubator was adjusted by modifying the N₂ content. Moreover, CO₂ concentration was maintained at 5%. Every 30 min, the O₂ concentration was changed from 1 to 21%. The cells were incubated for 6 days.

Yang L., Liu S., He Y., Gan L., Ni Q., Dai A., Mu C., Liu Q., Chen H., Lu H, & Sun R. (2024). Exosomes regulate SIRT3-related autophagy by delivering miR-421 to regulate macrophage polarization and participate in OSA-related NAFLD. Journal of Translational Medicine, 22, 475.

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Culturing AC16 Cardiomyocytes in DMEM/F12

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The AC16 cardiomyocytes were cultured in Dulbecco's Modified Eagle Medium/F12 medium (Gibco, USA), supplemented with 10% fetal calf serum (Gibco), and 1% penicillin-streptomycin solution (100x; Solarbio), then incubated in an incubator containing 5% CO₂ at 37°C.

Xie T., Yang Y., Gong K., Luo Y., Guo H., Liu R., Wang L., Tan Z., Luo J, & Xie L. (2022). Whole-Exome Sequencing Identifies a Novel Variant (c.1538T > C) of TNNI3K in Arrhythmogenic Right Ventricular Cardiomyopathy. Frontiers in Cardiovascular Medicine, 9, 843837.

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Culturing Ishikawa Endometrial Cancer Cells

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Ishikawa cells (a well-differentiated human endometrial adenocarcinoma line) obtained from American Type Cell Culture (Manassas, VA, USA) were maintained in a Dulbecco’s Modified Eagle Medium/F12 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco), 100 mg/mL streptomycin (Gibco), and 2 mM L-glutamine (Gibco). Cells were grown on Matrigel-coated cover glass (1:8 dilution, growth factor-reduced; Corning, Tewksbury, MA, USA) for further attachment assays.

Lee C.J., Hong S.H., Yoon M.J., Lee K.A., Ko J.J., Koo H.S., Kim J.H., Choi D.H., Kwon H, & Kang Y.J. (2020). Endometrial profilin 1: a key player in embryo-endometrial crosstalk. Clinical and Experimental Reproductive Medicine, 47(2), 114-121.

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