Dmem f12 10

Manufactured by Thermo Fisher Scientific

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DMEM-F12-10 is a cell culture medium developed by Thermo Fisher Scientific. It is a powdered formulation designed for the growth and maintenance of a variety of mammalian cell lines. The medium provides the necessary nutrients and components to support cell proliferation and differentiation in in vitro cell culture applications.

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DMEM/F12 (9007 citations) DMEM-10 (15 citations) DMEM/F12 10% FBS (9 citations) DMEM:F12 + 10% iFBS (2 citations)

7 protocols using dmem f12 10

Isolation and Culture of Murine Peritoneal Macrophages

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PMs were isolated according to a previously established method (Pineda-Torra et al., 2015 (link)). Briefly,
3% thioglycolate (Becton Dickinson, Sparks, MD, USA) in
phosphate-buffered saline (PBS) was intraperitoneally injected into C57BL/6 mice
(Orient Bio, Seongnam, Korea) at 1 mL per mouse. On day 3,
thioglycolate-injected mice were sacrificed to harvest peritoneal lavages. Cell
pellets were then acquired after centrifuging peritoneal lavages at 400×g
for 5 min at 4°C and resuspended in DMEM/F-12 (Thermo Fisher Scientific,
Waltham, MA, USA) medium supplemented with 10% (v/v) fetal bovine serum
and 1% penicillin/streptomycin (DMEM/F-12-10). After resuspension, cells
were plated into tissue culture plates and incubated in a humidified 5%
CO₂ atmosphere at 37°C. After overnight culture, adherent
cells were harvested and counted. The purity of PMs was validated by flow
cytometry analyses using anti-mouse CD11b (eBioscience, San Diego, CA, USA) and
anti-mouse F4/80 (BioLegend, San Diego, CA, USA) antibodies. More than
90% of cells were CD11b⁺F4/80⁺ cells
(data not shown). After isolation, PMs (2×10⁶ cell/well) were
seeded into a 12-well plate in 1 mL of DMEM/F-12-10. Heat killed WIKIM28 or
WIKIM50 (2×10⁷ CFU/mL) was co-cultured with PMs in a
humidified 5% CO₂ atmosphere at 37°C for 24 hours. As
controls, vehicle (PBS) was used to treat PMs without incubation with
probiotics.

Choi S.P., Park S.W., Kang S.J., Lim S.K., Kwon M.S., Choi H.J, & Chun T. (2023). Monitoring mRNA Expression Patterns in Macrophages in Response to Two Different Strains of Probiotics. Food Science of Animal Resources, 43(4), 703-711.

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Characterization of Alpha TC1-6 Cell Line

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The mycoplasma-free alpha TC1–6 cell line was purchased from ATCC (CRL-2934; Barcelona), and maintained in DMEM (ThermoFisher Scientific) supplemented with 10% FBS, 15 mM HEPES, 0.1 mM non-essential amino acids, 0.02% BSA, 2 g/l glucose, 1.5 g/l sodium bicarbonate and 5 µM beta mercaptoethanol (all purchased from Sigma-Aldrich). The proliferation of splenocytes isolated from mouse spleens was assessed after a 3-day culture in RPMI 1640 medium supplemented with 8% FBS, 20 mM l-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, and 1% penicillin/streptomycin (all from Invitrogen), in the presence or absence of the insulin peptide SLYQLENYCA. Cells were pulsed with [³H]-thymidine for the last 24 h of culture, harvested and lysed onto membranes prior to liquid scintillation counting using a Beckman Coulter LS 6500 counter. Mouse primary macrophages were isolated from the peritoneal cavity, and cultured in DMEM/F12–10 (ThermoFisher Scientific) supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (all purchased from Sigma-Aldrich). Cells were stimulated with 1 μg/ml LPS (in DMSO), in the absence or presence of 0.1, 1, or 10 μM BL001 for 24 h. The secretion of cytokines was measured in the culture medium by electrochemiluminescence technology from MesoScale Discovery (Rockville, USA), and RNA was extracted from cells.

Cobo-Vuilleumier N., Lorenzo P.I., Rodríguez N.G., Herrera Gómez I.D., Fuente-Martin E., López-Noriega L., Mellado-Gil J.M., Romero-Zerbo S.Y., Baquié M., Lachaud C.C., Stifter K., Perdomo G., Bugliani M., De Tata V., Bosco D., Parnaud G., Pozo D., Hmadcha A., Florido J.P., Toscano M.G., de Haan P., Schoonjans K., Sánchez Palazón L., Marchetti P., Schirmbeck R., Martín-Montalvo A., Meda P., Soria B., Bermúdez-Silva F.J., St-Onge L, & Gauthier B.R. (2018). LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus. Nature Communications, 9, 1488.

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Evaluating Nanotube Aggregation in Cell Culture

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To evaluate the aggregation of 053SNAP nanotubes conjugated with either SNAP-Surface® Alexa Fluor® 488 or SNAP-Cell® 647-SiR (New England Biolabs) in cell culture medium, the nanotube solution was diluted in Dulbecco's modified Eagle's medium with Nutrient Mixture F-12 (DMEM/F12-10, Gibco) supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS, Gibco) for a final concentration of 50 μg ml⁻¹ and incubated for 4 h in 37 °C, 5% CO₂. The solution was then imaged at 1 μm above the glass surface using a Leica TCS SP8 system with a 63×/1.4 NA oil-immersion objective for confocal imaging and a 100×/1.40 NA oil-immersion objective for stimulated emission depletion (STED) imaging. For STED imaging 775 nm depletion laser was used. Particle size from the micrographs was evaluated on ImageJ (FIJI) software.^{63 (link)}

Gabrielaitis D., Zitkute V., Saveikyte L., Labutyte G., Skapas M., Meskys R., Casaite V., Sasnauskiene A, & Neniskyte U. (2023). Nanotubes from bacteriophage tail sheath proteins: internalisation by cancer cells and macrophages. Nanoscale Advances, 5(14), 3705-3716.

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Cell Culture and Protein Expression Protocols

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MLE cells were cultured in Dulbecco’s Modified Eagle Medium-F12 (Gibco) supplemented with 10% fetal bovine serum (DMEM-F12-10). 293T cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum (DMEM-10). MRC5 cells were cultured in Eagle's Minimum Essential Medium (Gibco) supplemented with 10% fetal bovine serum (EMEM-10). For protein expression in MLE cells, nucleofection was used following Amaxa’s protocol. For protein overexpression in 293T cells, Fugene6HD transfection reagents were used following the manufacturer’s protocol. For protein expression in MRC5 cells, MRC-5 Cell Avalanche Transfection Reagent was used following the manufacturer’s protocol. Cells were treated with TGFβ at 0–2 ng/ml for 0–18 h. For FIEL1, PKCζ, or GSK3β knockdown studies in cells, scramble shRNA, FIEL1, PKCζ, or GSK3β shRNA were used to transfect cells for 48 h. For drug treatment, compounds were solubilized in DMSO before being added to the cells for up to 18 h. Cell lysates were prepared by brief sonication in 150 mM NaCl, 50 mM Tris, 1.0 mM EDTA, 2 mM dithiothreitol, 0.025% sodium azide, and 1 mM phenylmethylsulfonyl fluoride (Buffer A) at 4°C. For half-life study, MLE cells were exposed to cyclohexamide (40 µg/ml) in a time-dependent manner for up to 8 h. Cells were then collected and immunoblotted.

Lear T., McKelvey A.C., Rajbhandari S., Dunn S.R., Coon T.A., Connelly W., Zhao J.Y., Kass D.J., Zhang Y., Liu Y, & Chen B.B. (2016). Ubiquitin E3 ligase FIEL1 regulates fibrotic lung injury through SUMO-E3 ligase PIAS4. The Journal of Experimental Medicine, 213(6), 1029-1046.

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Murine Lung Cell Signaling Protocol

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Murine lung epithelial 12 cells (MLE) were obtained from ATCC (CRL-2110;
American Type Culture Collection, Manassas, VA) and cultured in
accordance with the manufacturer’s instructions. MLE cells were
cultured in DMEM-F12 (Gibco) with 10% FBS (DMEM-F12-10). MLE cells
were incubated with PSS (10 μM) for 1 h prior to LPS (1 μg/ml)
stimulation in an in vitro study. One h later, cells
were harvested for testing NF-κB signalling proteins by Western blot.
The total lung cells were obtained from the lung of mice according to
a protocol described before.^{17 (link)} Lung cells were then harvested for flow cytometry.

Zhao P., Liu G., Cui Y, & Sun X. (2019). Propylene glycol alginate sodium sulphate attenuates LPS-induced acute lung injury in a mouse model. Innate Immunity, 25(8), 513-521.

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Isolation of Rat Peritoneal Macrophages

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Macrophages were isolated from rat peritoneal in this study. Briefly, 5 ml of aseptic paraffin wax was injected into the peritoneal of 3-week-old SD rats. The rats were euthanized 4 days later, soaked with 70% alcohol, and then make a small incision along the midline with sterile scissors to expose the intact peritoneal wall. Then 10 ml of PBS was injected into the peritoneal from the incision, shake the abdomen and then aspirate fluid from peritoneum using the same syringe and needle. Centrifuge the aspirated fluid in a refrigerated centrifuge of 4°C for 10 min at 400 × g. Then resuspend the cells at the bottom of the tube with DMEM/F12-10 (Gibco) and seeded the cells to Transwell® polyester membranes (Corning 3460) with the concentration of 1 × 10⁶ cells/well. The plates were incubated at 37 °C for 2 hours and the medium was changed to remove the unadhered cells.

Zhang Q., Zhang Y., Yan M., Zhu K., Su Q., Pan J., Yang M., Zhou D, & Tan J. (2020). βig-h3 enhances chondrogenesis via promoting mesenchymal condensation in rat Achilles tendon heterotopic ossification model. Aging (Albany NY), 12(8), 7030-7041.

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Murine Lung Epithelial Cell Line Characterization

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Murine Lung Epithelial 12 cells (MLE) were from ATCC (CRL-2110) and cultured according to manufacturer’s instructions. The identity of the cell lines was monitored by microscope based morphology analysis and immunoblotting with multiple markers. The cell lines were checked for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, Switzerland). MLE cells were cultured in Dulbecco’s Modified Eagle Medium-F12 (Gibco) supplemented with 10% fetal bovine serum (DMEM-F12-10). For PPP1R11 overexpression in MLE cells, an Amaxa nucleofection kit was used following the manufacturer’s protocol. 24 hr later, cells were treated with doses of Pam3CSK4 up to 10 μg/ml. For PPP1R11 knockdown studies in MLE cells, scramble siRNA and PPP1R11 siRNA were used to transfect cells for 48 hr using electroporation. Cell lysates were prepared by brief sonication in 150 mM NaCl, 50 mM Tris, 1.0 mM EDTA, 2 mM dithiothreitol, 0.025% sodium azide, and 1 mM phenylmethylsulfonyl fluoride (Buffer A) at 4°C. For half-life study, MLE cells were exposed to cycloheximide (40 mg/ml) in a time dependent manner for up to 6 hr. Cells were then collected and immunoblotted. Protein densitometry was quantified through ImageJ and normalized to the zero time point for each set of condition.

McKelvey A.C., Lear T.B., Dunn S.R., Evankovich J., Londino J.D., Bednash J.S., Zhang Y., McVerry B.J., Liu Y, & Chen B.B. (2016). RING finger E3 ligase PPP1R11 regulates TLR2 signaling and innate immunity. eLife, 5, e18496.

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