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Dmem f12

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DMEM/F12 is a cell culture media formulation that supports the growth and maintenance of a variety of cell types, including mammalian and some insect cells. It is a basal medium that provides essential nutrients, vitamins, and salts required for cell proliferation and survival.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions) Dmem f12, by Merck Group (4 mentions) Lsm 880 confocal microscope, by Zeiss (4 mentions) Glutamax, by Thermo Fisher Scientific (4 mentions) Heparin, by Merck Group (3 mentions) Nahco3, by Thermo Fisher Scientific (3 mentions) N2 plus supplement, by R&D Systems (3 mentions) Hepes, by Thermo Fisher Scientific (3 mentions) Gentamicin sulfate, by Thermo Fisher Scientific (3 mentions) Fungizone, by Thermo Fisher Scientific (3 mentions) 4 well cell culture chamber slides, by Corning (2 mentions) Advanced dmem f12, by US Biological (2 mentions) Click it edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (2 mentions) 2 2 bipyridyl, by Merck Group (2 mentions) Mizoribine, by Merck Group (2 mentions) Dmem f12, by Thermo Fisher Scientific (2 mentions) Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions) Fibroblast growth factor (fgf), by Thermo Fisher Scientific (2 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)

27 protocols using dmem f12

1

Metabolite Extraction for Cellular Media

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Cells were plated in a 6-well plate at a density of 50,000 cells/well in DMEM/F-12 media supplemented with 10% FBS. 6 technical replicates were plated. 24 hrs after plating, media was changed to DMEM/F-12 (USBiological) containing 1.2 g/L sodium bicarbonate, 10 mM HEPES, 10% dialyzed FBS, 10 mM glucose, and 4.5 mM glutamine. Cells were incubated for another 24 hrs. Then, media was collected from each well. Next, 20 µL of each media sample was added to 180 µL of a 2:2:1 methanol:acetonitrile:water (−20°C) mixture. Then, samples were vortexed for 1 min and placed at −20°C for 1 hr. Next, samples were centrifuged at 16,000 g at 4°C for 10 min and supernatant was transferred to LC glass vials. Samples were stored at −80°C until LC/MS analysis (see below).
Sponagel J., Jones J.K., Frankfater C., Zhang S., Tung O., Cho K., Tinkum K.L., Gass H., Nunez E., Spitz D.R., Chinnaiyan P., Schaefer J., Patti G.J., Graham M.S., Mauguen A., Grkovski M., Dunphy M.P., Krebs S., Luo J., Rubin J.B, & Ippolito J.E. (2022). Sex Differences in Brain Tumor Glutamine Metabolism Reveal Sex-Specific Vulnerabilities to Treatment. Med (New York, N.Y.), 3(11), 792-811.e12.
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2

Cell Labeling and NMR Analysis

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Experimental design and data analysis and interpretation were consistent with previous publications60 (link),61 (link). Cells were plated in 150 mm dishes at a density of 300,000 cells/dish in DMEM/F-12 media supplemented with 10% FBS. 24 hours after plating, media was changed to DMEM/F-12 (USBiological) containing 1.2 g/L sodium bicarbonate, 10 mM HEPES, 10% dialyzed FBS, 10 mM glucose, and 4.5 mM [13C515N2]glutamine. Cells were incubated for 48 hrs. Then, cells were trypsinized and centrifuged at 200 g at 4°C for 5 min. Media was aspirated and the cell pellet was washed with ice cold 1X PBS. Washing was repeated two times and the cell pellet was stored at −80°C. Before NMR analysis the cell pellet was dried in a lyophilizer at −20°C. The total weight of the male and female dried cell pellet submitted for solid-state NMR analysis was 20 mg each and was accumulated over three sample preparations.
Sponagel J., Jones J.K., Frankfater C., Zhang S., Tung O., Cho K., Tinkum K.L., Gass H., Nunez E., Spitz D.R., Chinnaiyan P., Schaefer J., Patti G.J., Graham M.S., Mauguen A., Grkovski M., Dunphy M.P., Krebs S., Luo J., Rubin J.B, & Ippolito J.E. (2022). Sex Differences in Brain Tumor Glutamine Metabolism Reveal Sex-Specific Vulnerabilities to Treatment. Med (New York, N.Y.), 3(11), 792-811.e12.
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3

Amino Acid Supplemented DMEM/F12 Protocol

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Amino acid deficient DMEM/F12 was purchased (USBiological) and reconstituted as manufacturer’s instruction. Individual amino acids were purchased (Sigma) and prepared as a 100X stock solution which then used to make the corresponding final concentration in DMEM/F12 full media.
Vo V.T., Kim S., Hua T.N., Oh J, & Jeong Y. (2022). Iron commensalism of mesenchymal glioblastoma promotes ferroptosis susceptibility upon dopamine treatment. Communications Biology, 5, 593.
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4

Tryptophan Depletion Induces Chlamydia Knockdown

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HeLa cells were infected at a multiplicity of infection (moi) of 0.5 (t=0 hour post-infection (hpi)), and were maintained in complete DMEM for 14 h. The infected cells were then exposed to anhydrous tetracycline (aTc) at 5 nM for 4 h to induce knockdown of expression (t=14 hpi). Tryptophan depletion was performed at t=18 hpi by first washing cells with HBSS and then replacing complete DMEM with tryptophan-depleted DMEM-F12 (U.S. Biological Life Sciences). Treated cells were then returned to the tissue culture incubator for the remainder of the experimental time course. At t=22 hpi, samples were collected and fixed with freshly prepared 4% paraformaldehyde and permeabilized for 10 min with 0.1% Triton X-100 in PBS. Samples were immunostained with mouse antibody against the C. trachomatis major outer membrane protein (1:1000 dilution). Samples were incubated at 4C overnight with constant rocking. The next day, the primary antibody solution was removed and samples rinsed 3x with 1x PBS, and incubated with 1:1000 dilution of goat anti-mouse IgG conjugated with Alexa-488 for 1 h. Samples were rinsed and visualized by fluorescence microscopy. Images were processed and inclusion size measured using NIH ImageJ.
Chowdhury N.B., Pokorzynski N., Rucks E.A., Ouellette S.P., Carabeo R.A, & Saha R. (2023). Machine Learning and Metabolic Model Guided CRISPRi Reveals a Central Role for Phosphoglycerate Mutase in Chlamydia trachomatis Persistence. bioRxiv.
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5

Cultivation of Mouse Hepatocyte AML12 Cells

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Mouse hepatocyte AML12 cells were purchased from the Duke University Cell Culture Facility. AML12 cells were maintained in complete growth medium (CGM) which consisted of DMEM/F12 (US Biological) supplemented with 10% non-heat activated fetal bovine serum (FBS, GE Life Sciences Hyclone), penicillin and streptomycin (100 U/ml and 100 μg/ml, respectively, Gibco), 10 μg/ml insulin, 5 μg/ml transferrin, 7 ng/ml selenium (1:100 dilution of ITS, Millipore), and 100 nM dexamethasone (Sigma). Cells were grown in a humidified tissue culture incubator (Symphony, VWR) at 37 °C with 5% CO2. For experiments, cells were harvested using trypsin (0.25%) –ethylenediaminetetraacetic acid and seeded into 96-well plates (5×104 cells/well) in 200 μl of CGM and allowed to settle and grow for approximately 18 h at 37 °C with 5% CO2.
Mello D.F., Trevisan R., Rivera N., Geitner N.K., Di Giulio R.T., Wiesner M.R., Hsu-Kim H, & Meyer J.N. (2019). Caveats to the use of MTT, Neutral Red, Hoechst and Resazurin to measure silver nanoparticle cytotoxicity. Chemico-biological interactions, 315, 108868.
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6

Culturing Hippocampal HCN Cells

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HCN cells were obtained from the hippocampus of 8-week-old Sprague Dawley rats and maintained in coated culture dishes with poly-L-ornithine and laminin, as previously described [26 (link)]. HCN cells were cultured in a chemically defined serum-free media containing Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (12400–024, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with individually prepared N2 components and basic fibroblast growth factor (bFGF) (20 ng/mL; 100-18B-500, Peprotech, Cranbury, NJ, USA). Insulin withdrawal media were prepared by omitting insulin. To make glucose-low media, Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (D9807-02, USBiological, Salem, MA, USA) was supplemented with N2 components and bFGF. Then, D-glucose (Sigma-Aldrich, St. Louis, MO, USA) was added to a final concentration of 5 mM.
Jeong S.H., An H.K., Ha S, & Yu S.W. (2023). Distinct Signaling Pathways for Autophagy-Driven Cell Death and Survival in Adult Hippocampal Neural Stem Cells. International Journal of Molecular Sciences, 24(9), 8289.
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7

Quantifying PDO Proliferation in Serine-Glycine Depletion

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PDOs were seeded on 4-well cell culture chamber slides (Corning, 354114) at equal numbers. PDO media was replaced with vehicle or serine and glycine-free media the next day and incubated for 10 days. Serine and glycine-free Advanced DMEM/F12 was generated using amino acid, glucose, and pyruvate free DMEM/F12 (US Biological, D9807-11) supplemented with ethanolamine, glutathione, ascorbic acid, transferrin, and AlbuMAX II to the levels found in commercial Advanced DMEM/F12. PDO cultures were treated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 4h and fixed for 40 minutes with 4% PFA. The fixed PDOs were permeabilized with 0.5% Tx-100 for 20 minutes and labeled using the Click-iT EdU Alexa Fluor 488 imaging kit (ThermoFisher, C10337) according to manufacturer’s instructions. Labeled PDOs were imaged with a Zeiss LSM880 confocal microscope. The ratio of EdU+ cells was quantified in 16 to 25 organoids/group by counting the number of EdU+ cells per total number of cells. Statistical analysis was performed with Prism GraphPad. Student’s t-test was used to calculate p values.
Choi B.H., Rawat V., Högström J., Burns P.A., Conger K.O., Ozgurses M.E., Patel J.M., Mehta T.S., Warren A., Selfors L.M., Muranen T, & Coloff J.L. (2022). Lineage-specific silencing of PSAT1 induces serine auxotrophy and sensitivity to dietary serine starvation in luminal breast tumors. Cell reports, 38(3), 110278.
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8

Quantifying PDO Proliferation in Serine-Glycine Depletion

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PDOs were seeded on 4-well cell culture chamber slides (Corning, 354114) at equal numbers. PDO media was replaced with vehicle or serine and glycine-free media the next day and incubated for 10 days. Serine and glycine-free Advanced DMEM/F12 was generated using amino acid, glucose, and pyruvate free DMEM/F12 (US Biological, D9807-11) supplemented with ethanolamine, glutathione, ascorbic acid, transferrin, and AlbuMAX II to the levels found in commercial Advanced DMEM/F12. PDO cultures were treated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 4h and fixed for 40 minutes with 4% PFA. The fixed PDOs were permeabilized with 0.5% Tx-100 for 20 minutes and labeled using the Click-iT EdU Alexa Fluor 488 imaging kit (ThermoFisher, C10337) according to manufacturer’s instructions. Labeled PDOs were imaged with a Zeiss LSM880 confocal microscope. The ratio of EdU+ cells was quantified in 16 to 25 organoids/group by counting the number of EdU+ cells per total number of cells. Statistical analysis was performed with Prism GraphPad. Student’s t-test was used to calculate p values.
Choi B.H., Rawat V., Högström J., Burns P.A., Conger K.O., Ozgurses M.E., Patel J.M., Mehta T.S., Warren A., Selfors L.M., Muranen T, & Coloff J.L. (2022). Lineage-specific silencing of PSAT1 induces serine auxotrophy and sensitivity to dietary serine starvation in luminal breast tumors. Cell reports, 38(3), 110278.
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9

Culturing Panc1 Cells and Cancer Stem Cells

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The human Panc1 pancreatic adenocarcinoma cell line was grown in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, and 50 µg/ml gentamicin sulfate (Gibco, Thermo Fisher Scientific, Milan, Italy) at 37 °C with 5% CO 2 . Panc1 CSCs were generated as previously described [8] and cultured in CSC medium, [DMEM/F-12 (US biological Life Sciences) supplemented with 1g/l glucose, B27 (Gibco, Thermo Fisher Scientific), 1 µg/ml fungizone (Gibco, Thermo Fisher Scientific), 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific), 5 µg/ml heparin (Sigma-Aldrich), 20 ng/ml EGF (epidermal growth factor, Peprotech, London, UK), and 20 ng/ml FGF (fibroblast growth factor, Peprotech)] at 37 °C with 5% CO 2 . from the assay is proportional to the number of living cells in the well. Three independent experiments were performed for each condition and cell viability was reported as the percentage relative to control.
Marengo A., Forciniti S., Dando I., Dalla Pozza E., Stella B., Tsapis N., Yagoubi N., Fanelli G., Fattal E., Heeschen C., Palmieri M, & Arpicco S. (2019). Pancreatic cancer stem cell proliferation is strongly inhibited by diethyldithiocarbamate-copper complex loaded into hyaluronic acid decorated liposomes. Biochimica et biophysica acta. General subjects, 1863(1).
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10

Culturing Pancreatic Cancer Cell Lines

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Human pancreatic cancer cell lines PANC-1 and HPAC were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). PANC-1 and Pa8 cells were cultured in DMEM containing 4.5 g/L glucose, 100 units /ml penicillin and streptomycin, and 10% fetal bovine serum. PK1 cells were cultured in DMEM containing 4.5 g/L glucose, 100 units /ml penicillin and streptomycin, 10% fetal bovine serum, 10 ng/ml EGF (PeproTech), 10 ng/ml bFGF (PeproTech), and ITS (Insulin-Transferrin-Selenium; Invitrogen). HPAC cells were cultured in DMEM F-12 (USBiological) containing 3.151 g/L glucose, penicillin and streptomycin, and 10% fetal bovine serum. To study the effect of fructose replacement, glucose in the basal medium was replaced with 1g/L of fructose (Wako, Osaka, Japan).
Hsieh C.C., Shyr Y.M., Liao W.Y., Chen T.H., Wang S.E., Lu P.C., Lin P.Y., Chen Y.B., Mao W.Y., Han H.Y., Hsiao M., Yang W.B., Li W.S., Sher Y.P, & Shen C.N. (2016). Elevation of β-galactoside α2,6-sialyltransferase 1 in a fructose-responsive manner promotes pancreatic cancer metastasis. Oncotarget, 8(5), 7691-7709.
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