Ham s f12 media

Manufactured by Corning

Ham's F12 media is a cell culture medium used for the growth and maintenance of various cell types, including mammalian cells. It provides the necessary nutrients and growth factors required for optimal cell proliferation and survival. The medium is formulated to support a range of cell lines and is commonly used in applications such as cellular research, drug development, and biotechnology.

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Ham’s F12 (39 citations) F12 media (3 citations)

9 protocols using ham s f12 media

Isolation and Culture of Human Adipocytes

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Approximately 20 mL of tissue was dissected, avoiding blood vessels and fibrous areas. The tissue was then minced into 2-3 mm pieces and digested in 50 mL total volume of Ham’s F12 media (Corning) supplemented with 10% FBS, 1% penicillin/streptomycin, 10 mg/mL collagenase type I (Sigma Aldrich) and 10 ug/mL hyaluronidase (Sigma Aldrich). Tissue was digested for 1 hr at 37 °C on a rotator. The digested tissue was then centrifuged at 500 g for 10 min to separate the adipocyte fraction (floating layer) and stromal vascular fraction (pellet). Oil from lysed adipocytes was removed by aspiration and a wide orifice pipet tip was used to collect the adipocytes below the oil layer. Adipocytes were washed twice cultured in Ham’s F12 media (Corning) supplemented with 10% FBS, 1% penicillin/streptomycin (37 °C, 5% CO₂). Viral infections were performed within 24 h following adipocyte isolation.

Reiterer M., Rajan M., Gómez-Banoy N., Lau J.D., Gomez-Escobar L.G., Ma L., Gilani A., Alvarez-Mulett S., Sholle E.T., Chandar V., Bram Y., Hoffman K., Bhardwaj P., Piloco P., Rubio-Navarro A., Uhl S., Carrau L., Houhgton S., Redmond D., Shukla A.P., Goyal P., Brown K.A., tenOever B.R., Alonso L.C., Schwartz R.E., Schenck E.J., Safford M.M, & Lo J.C. (2021). Hyperglycemia in acute COVID-19 is characterized by insulin resistance and adipose tissue infectivity by SARS-CoV-2. Cell Metabolism, 33(11), 2174-2188.e5.

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Lentiviral Overexpression of KHK-C in HepG2 Cells

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Human hepatic HepG2 cells were cultured under standard conditions in 1:1 Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F-12 media (Corning) supplemented with 10% fetal bovine serum (FBS, Corning) and 1% penicillin-streptomycin in a 5% CO2-humidified cell culture incubator at 37°C. Lentiviral particles were purchased from Origene (MR204149L2V) containing the mouse KHK-C sequence fused to GFP. HepG2 cells were transduced with lentivirus at a MOI of 5 and media was changed every two days thereafter (https://star-protocols.cell.com/protocols/913). After 5 days, cells were sorted for GFP-intensity and only individual GFP+ cells were collected in 96-well plates and propagated for an additional 2–3 weeks. Similarly GFP tagged KHK-C was overexpressed in mouse AML-12 cells. KHK-C was quantified in these clones by both QPCR and western blot. Only the highest expressing clones were used for subsequent experiments.

Park S.H., Helsley R.N., Fadhul T., Willoughby J.L., Noetzli L., Tu H.C., Solheim M.H., Fujisaka S., Pan H., Dreyfuss J.M., Bons J., Rose J., King C.D., Schilling B., Lusis A.J., Pan C., Gupta M., Kulkarni R.N., Fitzgerald K., Kern P.A., Divanovic S., Kahn C.R, & Softic S. (2023). Fructose Induced KHK-C Increases ER Stress and Modulates Hepatic Transcriptome to Drive Liver Disease in Diet-Induced and Genetic Models of NAFLD.. bioRxiv.

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Culturing Pituitary and CHO Cell Lines

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The mouse corticotroph pituitary derived cell line AtT-20/D16v-F2 (ATCC® CRL-1795) was cultured and maintained in Dulbecco's Modified Eagle's Medium (DMEM) complemented with 10% FBS, 100 U/ml penicillin/streptomycin, 0.024 M of 2-(4(2-hydroxyethyl)-1-piperazine)-ethane sulfonic acid (HEPES), and maintained at 37°C and 5% CO₂, under sterile conditions. CHO-K1 cell line(ATCC® CCL-61) expressing recombinant human GHSR1a (GenBank accession number U60179) or GHSR1b (GenBank accession number U60181) were cultured and maintained in Ham's F12 media (Corning #10-080-CV) supplemented with 10% FBS, 2 mM L-Glutamine and 0.4 mg/mL Geneticin.

Ibáñez-Costa A., Gahete M.D., Rivero-Cortés E., Rincón-Fernández D., Nelson R., Beltrán M., de la Riva A., Japón M.A., Venegas-Moreno E., Gálvez M.Á., García-Arnés J.A., Soto-Moreno A., Morgan J., Tsomaia N., Culler M.D., Dieguez C., Castaño J.P, & Luque R.M. (2015). In1-ghrelin splicing variant is overexpressed in pituitary adenomas and increases their aggressive features. Scientific Reports, 5, 8714.

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Cell Culture Protocols for SUM159 and MRC5

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Human SUM159 cells were a gift from Dr. Carol Otey (UNC-Chapel Hill, Chapel Hill, NC) and were cultured in Ham’s F12 media (Corning) supplemented with 10% fetal bovine serum (FBS, Rocky Mountain Biologicals), 0.5 μg/ml hydrocortisone (Sigma), and 2.5 μg/ml insulin (Life Technologies). Human MRC5 fibroblasts were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning) supplemented with 10% FBS. All cells were grown at 37 °C and 5% CO₂. All experiments were conducted with early passage cells that were passaged no more than 15 times. Mycoplasma was tested regularly by staining with Hoechst 33342 (Anaspec).

Zinn A., Goicoechea S.M., Kreider-Letterman G., Maity D., Awadia S., Cedeno-Rosario L., Chen Y, & Garcia-Mata R. (2019). The small GTPase RhoG regulates microtubule-mediated focal adhesion disassembly. Scientific Reports, 9, 5163.

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Isolation and Culture of Mouse Tracheal Epithelial Cells

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HMW-HA (Yabro) a gift from IBSA Institut Biochimique, Lugano, Switzerland; LMW-HA was generated by sonication of Yabro. Hyaluronan dimers were generated by incubating 1ml (1.5mg/ml) of LMW-HA with 5μl (1U/μl) Hyaluronidase, streptomyces hyaluronlyticus nov. sp., ( Sigma-Aldrich 389561) at 37°C overnight.
Antibodies: αENaC (Invitrogen PA1-920A), βENaC (proteintech 14134-1-AP), γENaC (Invitrogen PA577797), CFTR (abcam ab2478), Na,K-ATPase-α (abcam ab76020), Na,K-ATPase-β1 (santa cruz sc-21713), CaSR ( Sigma-Aldrich C0493); actin (Sigma-Aldrich A5228). LysoTracker green (1 μM, ThermoFisher).
Media and compounds used for MTEC isolation and culture: Ham’s F12 media (Corning, MT10080CV); DMEM/F-12 medium (Corning , 11-320-033); Keratinocyte SFM (Gibco, 17005042); 100 X Antibiotic-Antimycotic (Pen/Strep/Fungiezone) Solution (Cytiva HyClone, SV3007901); Pronase (Roche, 10165921001), DNAse I (Sigma Aldrich, 10104159001); Murine EGF (Sigma, E5160); Bovine Pituitary Extract (Gibco, 13028014); Isoproterenol (Sigma, I-6504); Y-27632 (Selleck Chemical LLC, 129830-38-2); DAPT (Sigma, D5942); 5% Fetal Calf Serum (HyClone, SH30071.03); ITS-G (Gibco, 41400045); Cholera Toxin (Sigma, C8052); Retinoic Acid (Tocris Bioscience, 069550); BSA (Gibco, 15260037); Transwell^™ Multiple Well Plate with Permeable Polyester Membrane Inserts (Corning, 07-200-154).

Lazrak A., Song W., Yu Z., Zhang S., Nellore A., Hoopes C.W., Woodworth B.A, & Matalon S. (2023). Low Molecular Weight Hyaluronan Inhibits Lung Epithelial Ion Channels by Activating the Calcium-Sensing Receptor. Matrix biology : journal of the International Society for Matrix Biology, 116, 67-84.

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Maintaining Kv2.1-CHO and BFP-CHO Cell Lines

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All CHO-K1 cell lines were maintained in polystyrene dishes (BioLite, Thermo) at 37 °C in a 5% CO₂ atmosphere in Ham’s F-12 media (Corning) containing 10% v/v FBS (Gibco), and penicillin–streptomycin (Sigma). A CHO-K1 cell line stably transfected with rat Kv2.1 (Kv2.1-CHO cells)^{56 (link)} was cultured with 1 μg/mL blasticidin and 25 μg/mL zeocin to retain stably transfected vectors. To induce expression of Kv2.1, minocycline (Enzo Life Sciences) from a stock of 2 mg/ml in 70% EtOH was added to cell culture media to a final concentration 1 μg/mL. Time of incubation with minocycline was chosen to induce a desirable amount of channel expression: 1.5 – 2 h for K⁺ conductance experiments, ~48 h for imaging and voltage clamp spectroscopy experiments. The CHO-K1 cell line stably transfected with BFP (BFP-CHO cells)^{23 (link)} expresses the blue fluorescent protein variant EBFP2 with a nuclear localization sequence.^{57 (link)} BFP-CHO cells were maintained in media containing 100 μg/mL of G418.

Fletcher-Taylor S., Thapa P., Sepela R.J., Kaakati R., Yarov-Yarovoy V., Sack J.T, & Cohen B.E. (2020). Distinguishing Potassium Channel Resting State Conformations in Live Cells with Environment-Sensitive Fluorescence. ACS chemical neuroscience, 11(15), 2316-2326.

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Cell Culture Methodology for COS1 and CHO/hIRc

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COS1 cells (ATCC) were maintained in a humidified atmosphere of 5% CO₂ at 37 °C and cultured in DMEM media (Invitrogen) supplemented with 10% FBS (Invitrogen), 1% penicillinestreptomycin (Invitrogen), 1% GlutaMax (Invitrogen), and 1% non-essential amino acids (Invitrogen). CHO cells overexpressing the human insulin receptor (CHO/hIRc) were a kind gift from Dr. Michael L. Tremblay. CHO/hIRc cells were maintained in a humidified atmosphere of 5% CO₂ at 37 °C and cultured in Ham's F-12 media (Corning) supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% non-essential amino acids. For insulin treatment, cells were serum starved for 16 h prior to experimentation.

Garcia F.J, & Carroll K.S. (2014). Redox-based probes as tools to monitor oxidized protein tyrosine phosphatases in living cells. European journal of medicinal chemistry, 0, 28-33.

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Transient Transfection of CHO Cells

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Chinese hamster ovary (CHO) cells (ATCC Cat# CCL-61, RRID:CVCL_0214; American Type Culture Collection) were cultured in Ham’s F12 media (10-080, Corning/Cellgro) supplemented with 10% fetal bovine serum (Genesee Scientific Cat#25-514H). Transient transfection assays were carried out using the Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc., Cat#11668019). Cells were grown to 90% confluency, and transfections were performed according to the manufacturer’s instructions and according to the protocols outlined in.
^{24 (link)}^,^{25 (link)} The chick
N-cadherin-expressing (pCIG-N-cadherin) and empty (pCIG) vectors were gifts from Dr. Marianne Bronner (California Institute of Technology).

Halmi C.A., Wu C.Y, & Taneyhill L.A. (2022). Neural crest cell-placodal neuron interactions are mediated by Cadherin-7 and N-cadherin during early chick trigeminal ganglion assembly. F1000Research, 11, 741.

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NF-κB Activation Assay in Lung Cancer Cells

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NCI-H520 cells were from American Type Culture Collection. LC-2/ad cells were from RIKEN BRC. Cells were maintained in a humidified atmosphere with 5% carbon dioxide using Ham’s F-12 media (Cellgro, Manassas, Va) or Roswell Park Memorial Institute Media 1640 (Gibco, Grand Island, NY), respectively, with 10% fetal bovine serum (FBS). Tumor necrosis factor-alpha (TNF-α) was used to induce NF-κB activation. It was dissolved in phosphate buffered saline (PBS) and used at a concentration of 20 ng/mL. Antibodies for phosphorylated and total NF-κB and GAPDH were obtained from Cell Signaling Technology, Inc (Beverly, Mass). Antibody for S100A7 was obtained from Abcam (Cambridge, Mass).

Liu G., Wu Q., Liu G., Song X, & Zhang J. (2015). Psoriasin (S100A7) is a novel biomarker for lung squamous cell carcinoma in humans. Cancer Cell International, 15, 18.

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