F10 medium

Manufactured by Thermo Fisher Scientific

Sourced in United States

F10 medium is a cell culture medium designed to support the growth and maintenance of a wide range of cell types. It provides a balanced formulation of nutrients, vitamins, and other components necessary for cell proliferation and survival.

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Ham’s F10 medium (49 citations) HAM-F10 medium (4 citations) Ham’s F10 nutrient mixture medium (3 citations) DMEM/HAM F10 (1:1) medium (3 citations) F10 basal medium (1 citations)

23 protocols using f10 medium

Dissociation and Immunophenotyping of Limb Muscle

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Preparation of limb muscle for flow cytometric analysis was adapted from Liu et al. [28 (link)]. In short, dissected tissue was minced thoroughly to small pieces in F10 medium (Lonza) containing collagenase II (750 U/ml; Fisher Scientific) using scalpels. Minced tissue was dissociated for 70 min in F10 medium containing 750 U/ml collagenase II), then for 30 min in F10 medium containing collagenase II (100 U/ml) and dispase (1.1 U/ml; Fisher Scientific). Cell suspensions were filtered over a 40 μM cell strainer (Falcon) and a small sample was retrieved to determine total mononuclear cell count using a hematocytometer. The cell suspensions were stained with CD31-APC (1:100), CD45-APC (1:100), Sca1-FITC (1:100) and Vcam-biotin (1:50) primary antibodies. Vcam was visualized using streptavidin-PECY7 (1:100). All antibodies for flowcytometry were derived from BD Biosciences. Cell viability was determined by staining with 1 μg/ml Hoechst 33258 (Sigma). Samples were analyzed on a BD-ARIAIII (BD biosciences).

Schaaf G.J., van Gestel T.J., in ‘t Groen S.L., de Jong B., Boomaars B., Tarallo A., Cardone M., Parenti G., van der Ploeg A.T, & Pijnappel W.W. (2018). Satellite cells maintain regenerative capacity but fail to repair disease-associated muscle damage in mice with Pompe disease. Acta Neuropathologica Communications, 6, 119.

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Culturing Rat and Murine Pituitary Tumor Cells

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Rat pituitary tumoral lactotroph MMQ cells (ATCC CRL-10609) were cultured in RPMI medium (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 15% horse serum (HS), 2.5% fetal bovine serum (FBS), 2 mM glutamine and antibiotics (Gibco, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). Murine pituitary tumoral corticotroph AtT-20 cells (ATCC CCL-89) were grown in F-10 medium (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, 2 mM glutamine and antibiotics.

Mangili F., Treppiedi D., Catalano R., Marra G., Di Muro G., Spada A., Arosio M., Peverelli E, & Mantovani G. (2021). A Novel Mechanism Regulating Dopamine Receptor Type 2 Signal Transduction in Pituitary Tumoral Cells: The Role of cAMP/PKA-Induced Filamin A Phosphorylation. Frontiers in Endocrinology, 11, 611752.

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Tumor Disaggregation and Cell Culture

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Fresh tumor samples (verified from frozen sections), aseptically collected in the operating room, were minced with scissors in a petri dish. The tumor pieces were then disaggregated for 4 h at 37°C in 0.5% type IV collagenase (Sigma-Aldrich, St Louis, MO, USA) in F10 medium (Life Technologies, Carlsbad, CA, US). Then, the cells were pelleted and resuspended in the medium with 15% FBS. Cells were used for experiments after being cultured for 1 week.

Sun Y.C., Wang J., Guo C.C., Sai K., Wang J., Chen F.R., Yang Q.Y., Chen Y.S., Wang J., To T.S., Zhang Z.P., Mu Y.G, & Chen Z.P. (2014). MiR-181b sensitizes glioma cells to teniposide by targeting MDM2. BMC Cancer, 14, 611.

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Myoblast and Stem Cell Culture Protocols

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Myoblasts were cultured in F10 medium (Life Technologies, Carlsbad, CA) supplemented with 20% fetal bovine serum (Thermo Scientific, Waltham, MA), 10 ng bFGF (Life Technologies), 1 µM dexamethasone (Sigma) and 1 × penicillin/streptomycin (Life Technologies). For differentiation the proliferating myoblasts were plated at a seeding density of 70,000 cells/cm² and cultured with or without 1 mM EGTA in DMEM: F12 (1:1, Life Technologies) supplemented with 20% KOSR (Life Technologies) and 1 × penicillin/streptomycin (Life Technologies) for 48–72 h.
Human stem cells were cultured on irradiated mouse embryonic fibroblasts in media containing F12/DMEM (Life Technologies), 20% knockout serum replacer (Life Technologies), 1 × penicillin/streptomycin (Life Technologies), 1% 100 mM sodium pyruvate (Life Technologies), 1 × non-essential amino acid (Life Technologies), 0.1% 0.1 M β-mercaptoethanol (Sigma) and 2 ng/ml FGF-2 (Life Technologies). Stem cell colonies were separated from irradiated mouse embryonic fibroblast layers using dispase (Life Technologies) and seeded to matrigel-coated dishes in mTESR1 media (Stemcell Technologies) and cultured at 37 °C in 5% oxygen and 5% CO₂.

Haynes P., Bomsztyk K, & Miller D.G. (2018). Sporadic DUX4 expression in FSHD myocytes is associated with incomplete repression by the PRC2 complex and gain of H3K9 acetylation on the contracted D4Z4 allele. Epigenetics & Chromatin, 11, 47.

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Preparation and Use of Engineered Cell Lines

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The human embryonic kidney HEK293 cell line was previously transfected with the mouse IL4I1 cDNA cloned in pcDNA3.1 mychis (Invitrogen) or in pEGFP-N1 or with the control vector as described in [8] .
The monocytic THP1-IL4I1 cell line, described in Marquet et al. [14] , was used in T-cell cocultures after a 200 Gy irradiation. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll centrifugation (UNI-SEPmaxi, NOVAmed) of healthy donor cytaphereses from the French Blood Bank (EFS, Créteil). Human experiments were performed in F10 medium (Life Technologies) supplemented with 10% human AB serum from GE Healthcare.
Mouse experiments were performed in RPMI supplemented with 10% FCS from ID-bio and 50 μM β-mercaptoethanol. All media were supplemented with penicillin (100 UI/mL) and streptomycin (100 mg/mL) from Life Technologies. In Figure 5, RPMI without Phe and glutathione was custom made by Clinisciences. Phe, H 2 O 2 , and glutathione were from Sigma.
Recombinant His-tagged IL4I1 was partially purified using Ni-agarose bead (Qiagen, Courtaboeuf, France) as previously described [8] . Recombinant cytokines were from R&D Systems (IL-2 and IL-4), Peprotech (IL-1β, IL-6, IL-23, and TGF-β1), and Immunotools (IL-12).

Cousin C., Aubatin A., Le Gouvello S., Apetoh L., Castellano F, & Molinier-Frenkel V. (2015). The immunosuppressive enzyme IL4I1 promotes FoxP3(+) regulatory T lymphocyte differentiation. European journal of immunology, 45(6).

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Maintenance of Human Cell Lines

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The human androgen-independent cell line PC-3 (American Type Culture Collection, USA) were maintained in DMEM (PC-3) media (Invitrogen, France) supplemented with 10% fetal bovine serum (FBS). The rat colon carcinoma cancer cell line REG was provided by Dr Carmen Garrido (INSERM U866, Dijon, France) and maintained in F10 medium (Invitrogen) supplemented with 10% FBS. The Human Embryonic Kidney cell line HEK293T (American Type Culture Collection, USA) was maintained in Dulbecco's Modified Eagle's Medium (Invitrogen), supplemented with 10% FBS. All cell lines were cultivated at 37°C in 5% CO₂. PC-3DR-docetaxel resistant cell line [19 (link)] was kindly provided by Dr Martin Gleave (Vancouver Prostate Cancer Center) and was maintained in culture as previously described.

Ziouziou H., Andrieu C., Laurini E., Karaki S., Fermeglia M., Oueslati R., Taieb D., Camplo M., Siri O., Pricl S., Katsogiannou M, & Rocchi P. (2017). Targeting Hsp27/eIF4E interaction with phenazine compound: a promising alternative for castration-resistant prostate cancer treatment. Oncotarget, 8(44), 77317-77329.

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Optimized Hybrid Cell Line Culturing for Genetic Research

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The NIGMS human/rodent somatic cell hybrid mapping panel #2, version 3 was from Coriell Cell Repositories (Camden, NJ), in which human chrs 1, 16, 17, 20, and 21 hybrids are from mice, whereas the others are from Chinese hamsters. Mouse somatic cell hybrids containing human chrs 4, 10, 13, 14, 15, or 21 (catalog number GM11687, 11688, 11689, 10479, 11715, or 08854, respectively, from Coriell Cell Repositories) were grown in DMEM medium supplemented with 10% FBS, penicillin/streptomycin, and 2 mM L-glutamine. Normal, FSHD1 and FSHD2 myoblasts were grown in F-10 medium (Invitrogen, Carlsbad, CA) supplemented with 20% FBS, 10 ng/ml bFGF (Invitrogen), and 20 ng/ml dexamethasone sodium phosphate (Sigma–Aldrich, St. Louis, MO) except FSHD1 myoblasts (GM17731 and GM17940 from Coriell Cell Repositories) were grown in F-10 medium supplemented with 15% FBS. KD3 human myoblasts immortalized by the expression of telomerase, cyclin D1, and mutated cyclin-dependent kinase 4 are grown as previously described [Shiomi et al., 2011 (link)]. Control (NFGr) and FSHD2 (KII-II) fibroblasts were grown in DMEM/F-12 (1:1) supplemented with 10% FBS, 2 mM GlutaMAX-I (Invitrogen-Gibco, Carlsbad, CA), 10 mM HEPES buffer (Invitrogen-Gibco), and 1 mM sodium pyruvate (Invitrogen-Gibco). Control and patient cells used for ChIP analysis are shown in Supp. Table S1.

Zeng W., Chen Y.Y., Newkirk D.A., Wu B., Balog J., Kong X., Ball AR J.r., Zanotti S., Tawil R., Hashimoto N., Mortazavi A., van der Maarel S.M, & Yokomori K. (2014). Genetic and Epigenetic Characteristics of FSHD-Associated 4q and 10q D4Z4 that are Distinct from Non-4q/10q D4Z4 Homologs. Human mutation, 35(8), 998-1010.

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Isolation and Differentiation of Primary Myoblasts

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Primary myoblasts were isolated from the forelimbs and hindlimbs of three or four 5-day-old littermates57 (link). The muscles were dissected and minced, disaggregated enzymatically in 4 ml PBS containing 1.5 U/mL dispase II and 1.4 U/mL collagenase D (Roche, Grenzacherstrasse, Basel, Switzerland), and triturated with a 10-ml pipette every 5 min for 20 min at 37 °C. The cells were filtered through a 70-μm mesh (BD Bioscience, San Jose, CA, USA) and centrifuged at 1000 × g for 5 min. The cell pellet was dissociated in 10 mL F10 medium (Invitrogen, Life Technologies) supplemented with 10 ng/mL basic fibroblast growth factor (PeproTech; Rocky Hill, NJ, USA) and 10% cosmic calf serum (GE Healthcare). Finally, the cells were pre-plated twice on non-collagen coated plates for 1 h to deplete fibroblasts, which generally adhere faster than myoblasts. For differentiation, the primary myoblasts obtained were cultured to 75% confluence in DMEM containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 5% horse serum (Invitrogen, Life Technologies).

Kim N., Nam M., Kang M.S., Lee J.O., Lee Y.W., Hwang G.S, & Kim H.S. (2017). Piperine regulates UCP1 through the AMPK pathway by generating intracellular lactate production in muscle cells. Scientific Reports, 7, 41066.

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Fibroblast Responses to Hypoxia and Oxidative Stress

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Human dermal primary fibroblasts isolated from foreskin samples were cultured in F10 medium (Gibco, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS), 0.5% penicillin and 2% glutamine.
For hypoxic conditions, fibroblasts were maintained for 24 h (for RNA analysis) or 48 h (for protein analysis) in 1% O 2 by using a Hypoxia Incubator Chamber (Stemcell Technologies, Grenoble, France).
The oxidative stress was produced by adding hydrogen peroxide
to cultured fibroblasts at different doses (0-200 mM) for the indicated times. Cell proliferation was measured by colorimetric immunoassay based on BrdU incorporation (Roche Diagnostics). Total cellular glutathione was detected after 4 h incubation by means of the GSH + GSSG/GSH Assay Kit (Abcam). Chronic hyperglycemic conditions were obtained by culturing fibroblasts for three weeks in commercially available Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich) containing 25 mM or 5.5 mM D-glucose. The high glucose and low glucose DMEM had similar osmolarity.

Baldini E., Testa E., Voellenkle C., De Domenico E., Cianfarani F., Martelli F., Ulisse S, & Odorisio T. (2020). Dysregulation of microRNA expression in diabetic skin. Journal of dermatological science, 98(3).

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Isolation and Culture of Primary Myoblasts

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Primary myoblasts were isolated from the forelimbs and hindlimbs of 3–4 5-day-old littermates (Bois & Grosveld 2003 (link)). The muscles were dissected and minced, were disaggregated enzymatically in 4 ml PBS containing 1.5 U/ml dispase II and 1.4 U/ml collagenase D (Roche), and were triturated with a 10-ml pipette every 5 min for 20 min at 37 °C. The cells were filtered through a 70-μm mesh (BD Bioscience, CA, USA) and were centrifuged at 1000× g for 5 min. The cell pellet was dissociated in 10 ml F10 medium (Invitrogen, Life Technologies) supplemented with 10 ng/ml basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, USA) and 10% cosmic calf serum (referred to as growth medium 1; GE Healthcare). Finally, the cells were pre-plated twice on non-collagen coated plates for 1 h to deplete fibroblasts that generally adhere faster than myoblasts. For differentiation, the primary myoblasts obtained were cultured to 75% confluence in DMEM containing antibiotics and 5% horse serum (Invitrogen, Life Technologies).

Kim N., Lee J.O., Lee H.J., Lee Y.W., Kim H.I., Kim S.J., Park S.H., Lee C.S., Ryoo S.W., Hwang G.S, & Kim H.S. (2016). AMPK, a metabolic sensor, is involved in isoeugenol-induced glucose uptake in muscle cells. The Journal of Endocrinology, 228(2), 105-114.

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