Dmem f12 without phenol red

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DMEM/F12 without phenol red is a cell culture medium formulation that is commonly used for the in vitro cultivation of various cell types. It is a serum-free, chemically-defined medium that provides the necessary nutrients and growth factors for the maintenance and proliferation of cells. The absence of phenol red, a pH indicator, makes this formulation suitable for applications where spectrophotometric measurements or other light-sensitive experiments are required.

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38 protocols using dmem f12 without phenol red

Isolation and Culture of Endometrial Stromal Cells

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The primary tissue samples and OESC and CESC cells analyzed in this study are in S1 Table. Cryopreserved endometrial tissue obtained by diagnostic laparoscopy was minced and incubated with collagenase (Sigma-Aldrich, St. Louis, MO) at 37°C for 10 min., followed by filtration, as previously described [22 (link)]. This method produces 95–99% pure stromal cells. The purity of these stromal cells at passage one was evaluated by immunofluorescence analysis using antibodies against vimentin (stromal cell marker), cytokeratin7 (epithelial cell marker) and CD45 (leukocyte marker), which showed that all cultures were 98–99% pure stromal mesenchymal cells. The cells were then cultured as previously described [23 (link)]. Briefly, the cells were cultured on fibronectin-collagen (Gibco, Grand Island, NY) coated dishes in DMEM-F12 without phenol red (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine (Gibco) and 1% antibiotics–antimycotic (Gibco) up to passage 3. To exclude influences of the serum derived steroid hormones on DNA methylation and expression in the cultures, the cells were grown in culture medium containing 10% charcoal stripped fetal bovine serum (CS-FBS; Gibco).

Yotova I., Hsu E., Do C., Gaba A., Sczabolcs M., Dekan S., Kenner L., Wenzl R, & Tycko B. (2017). Epigenetic Alterations Affecting Transcription Factors and Signaling Pathways in Stromal Cells of Endometriosis. PLoS ONE, 12(1), e0170859.

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TGF-β1 Induced Fibrosis in ESCs

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HESCs and primary ESCs were cultured in DMEM/F-12 without phenol red (Gibco, Waltham, MA, USA), supplemented with 10% charcoal-stripped FBS (Biological Industries, Israel). The cells were cultured at 37 °C in a 5% CO₂ atmosphere. To avoid serum interference, the cells were starved in DMEM/F12 containing 0.1% FBS for 12 h before adding TGF-β1 (100-21C, Peprotech, Rocky Hill, NJ, USA) to induce cell fibrosis. The PI3K inhibitor LY294002 (15 μM, MCE, USA) was pretreated for 1 h before use.

Guo Z., Wang Y., Wen X., Xu X, & Yan L. (2022). β-Klotho Promotes the Development of Intrauterine Adhesions via the PI3K/AKT Signaling Pathway. International Journal of Molecular Sciences, 23(19), 11294.

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Isolation and Decidualization of Endometrial Cells

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Collected HESCs were isolated, cultured, and maintained as described previously^{20 (link),55 (link)}. We obtained the endometrial samples in Dulbecco’s modified Eagle’s medium-Ham’s (DMEM) F-12 (Nacalai Tesque, Kyoto, Japan) containing 1% (v/v) antibiotic solution, minced finely and digested enzymatically with 5 mg collagenase (5 mg/100 μl) (Sigma-Aldrich, Saint Louis, USA) and deoxyribonuclease (DNase) type I (100 μg/μl) (Roche Applied Science, Mannheim, Germany) for 1 h at 37 °C. After centrifugation, the cells were suspended in DMEM/F12 culture media containing 10% (v/v) dextran-coated charcoal (DCC) treated fetal bovine serum, 1% antibiotic solution, 1% (v/v) l-glutamine, 0.2% (v/v) insulin and 1 nM estradiol (all Sigma-Aldrich). Endometrial cells were cultured until confluence in 75 cm² culture flasks at 37 °C in 5% carbon dioxide and then passaged once or twice. Cells were discarded after passage 3. In decidualization experiments, confluent monolayers were maintained in DMEM/F12 without phenol red (GIBCO, life technologies, Grand Island, USA) containing 2% (v/v) DCC-FBS and treated with 0.5 mM 8-bromo-cAMP, 1 μM P4 in combination with or without 100 µM resveratrol (all Sigma-Aldrich).

Ochiai A., Kuroda K., Ozaki R., Ikemoto Y., Murakami K., Muter J., Matsumoto A., Itakura A., Brosens J.J, & Takeda S. (2019). Resveratrol inhibits decidualization by accelerating downregulation of the CRABP2-RAR pathway in differentiating human endometrial stromal cells. Cell Death & Disease, 10(4), 276.

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Visualizing HCV Entry in CD81-GFP Organoids

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Huh-7 cells expressing CD81-GFP were mixed with Matrigel; 400 mL of the solution was seeded into a 48-well plate and maintained as described. After 7 days, organoids were extracted from Matrigel and resuspended in 1 mL imaging media containing DMEM-F12 without phenol red (GIBCO) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 0.1 mM nonessential amino acids (GIBCO), 1% penicillin-streptomycin (GIBCO), and 50 mM HEPES (GIBCO). Organoids were then replated onto collagen-treated 35 mm imaging dishes with interlocking lids (Ibidi), 200 mL of the cell solution per plate. Imaging media was added to the dishes, and organoids were allowed to adhere for 6–8 hr. Just prior to imaging, DiD-labeled HCV was added to imaging dishes. Dishes were incubated on ice for 1 hr, then placed on an enclosed stage heated to 37°C on a Leica SP5 Tandem Scanner Spectral 2-Photon confocal microscope.
Cells were visualized using a 63x NA 1.4 oil-immersion objective. CD81-GFP was imaged with an Argon laser and a HyD detector set for the 495–535 nm wavelength range; DiD was imaged with a HeNe laser and a HyD detector using the 600–670 nm wavelength range. Videos were acquired through sequential exposures every 30 s up to 90 min post-temperatures shift. Images were processed using ImageJ.

Baktash Y., Madhav A., Coller K.E, & Randall G. (2018). Single Particle Imaging of Polarized Hepatoma Organoids upon Hepatitis C Virus Infection Reveals an Ordered and Sequential Entry Process. Cell host & microbe, 23(3), 382-394.e5.

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Establishing HK-2-hapoM Stable Cell Line

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HK-2 cells (ATCC, CRL-2190) were cultured on collagen coated wells (Type 1 Collagen, 5 μg/cm² overnight, Sigma), in DMEM/F12 without phenol red (21041–025, Gibco) supplemented with 1 × GlutaMAX (Gibco), 1 × P/S (Gibco), 20 mM Hepes (Gibco), 25 ng/mL Hydrocortizone (H6909, Sigma) and 1 × ITS supplement (41400045, Gibco). 200 μg/mL Zeocin (45–0430, Invitrogen) was added for selection.
A stable cell line overexpressing human apoM (HK-2-hapoM^TG) was established by transfecting the HK-2 cells with full length human apoM under the control of the human cytomegalovirus (CMV) promotor using the Flp-In system (Invitrogen). For experiments, cells were plated on collagen coated polycarbonate transwells (3401, Costar) and cultured until confluency. At day 4 after confluency was reached, cells were washed ones with PBS and the medium was changed to medium without FBS but with 0.05 mM BSA (A7030, Sigma).

Bisgaard L.S., Christensen P.M., Oh J., Torta F., Füchtbauer E.M., Nielsen L.B, & Christoffersen C. (2024). Kidney derived apolipoprotein M and its role in acute kidney injury. Frontiers in Pharmacology, 15, 1328259.

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Rat Ameloblastic Cell Line HAT-7 Culture

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The rat ameloblastic cell line HAT-7,^{41 (link)} established from the cervical loop of rat incisors, was provided by Pr. H. Harada (Iwate Medical University, Japan) and cultured as previously described.^{25 (link)} Briefly, cells were cultured in Dulbecco’s Modified Eagle Medium, Nutrient Mixture F12 (DMEM/F-12; GIBCO BRL), supplemented with 10% fetal bovine serum (FBS),

100 units per milliliter

penicillin, and

100 units per milliliter

streptomycin. Twelve hours before the treatments,

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cells/well (

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35 millimeters

) were seeded in DMEM/F-12 without phenol red (GIBCO BRL) containing 10% charcoal-treated FBS,

100 international units per milliliter

penicillin and

100 micrograms per milliliter

streptomycin. Treated cells were cultured in the same medium without FBS, containing

10 begin superscript negative 10 end superscript

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M mono(2-ethylhexyl) phthalate (MEHP; Sigma-Aldrich) (which is a DEHP metabolite) or an equivalent volume of 0.1% dimethyl sulfoxide (vehicle), for 48 h.

Bui A.T., Houari S., Loiodice S., Bazin D., Sadoine J., Roubier N., Vennat E., Tran T.T., Berdal A., Ricort J.M., Mhaouty-Kodja S, & Babajko S. (2022). Use of Dental Defects Associated with Low-Dose di(2-Ethylhexyl)Phthalate as an Early Marker of Exposure to Environmental Toxicants. Environmental Health Perspectives, 130(6), 067003.

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Culturing Mouse Neural Stem Cells

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Briefly, mouse NSCs were dissected from cerebral cortices of C57BL/6 J mouse fetal brains (E12.5) and cultured in poly-D-lysine (5 μg/ml, 2 h 37 °C) and laminin (5 μg/ml 37 °C, 4 h 37 °C) precoated dishes⁷⁷ and have subsequently been maintained in culture as a stable cell line. NSCs were grown with a medium prepared by mixing equal parts of DMEM F12 (without Phenol Red, Gibco) and Neural Basal Media (Gibco), Glutamax (1%), N2 and B27 supplements (Gibco), sodium pyruvate (1 mM), non-essential amino acids (0.1 mM), Heparin (2 mg/l), Hepes (5 mM), bovine serum albumin (25 mg/l) and β-mercaptoethanol (0.01 mM)^{23 (link)}. Fresh recombinant human Epidermal Growth Factor (EGF) (R&D systems) and Fibroblast Growth Factor (FGF) (Invitrogen) to 20 ng/ml and 10 ng/ml final concentrations respectively were added to the media. TGFβ (Millipore) was used at a final concentration of 5 ng/ml. Human HEK293T cells were cultured in DMEM supplemented with 10% of fetal bovine serum (Gibco) and 1% of Penicillin/Streptomycin^{78 (link)}.

Vicioso-Mantis M., Fueyo R., Navarro C., Cruz-Molina S., van Ijcken W.F., Rebollo E., Rada-Iglesias Á, & Martínez-Balbás M.A. (2022). JMJD3 intrinsically disordered region links the 3D-genome structure to TGFβ-dependent transcription activation. Nature Communications, 13, 3263.

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Isolation and Characterization of Primary Endometrial Stromal Cells

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Endometrial tissue obtained from control patients by curettage or from ectopic lesions from endometriosis patients by laparoscopic surgery was used to make primary endometrial stromal cells as previously described [57 (link)]. Briefly, the tissue was minced and incubated with collagenase (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 10 min, filtered and then cultured further, as previously documented [58 (link)]. The cells were cultured on fibronectin–collagen-(Gibco, Grand Island, NY, USA) coated dishes in DMEM-F12 without phenol red (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine (Gibco) and 1% antibiotics–antimycotic (Gibco) up until a maximum of passage 7 in a 37 °C CO₂-humified incubator. The purity of these stromal cells was evaluated using immunofluorescence analysis with antibodies against vimentin (stromal cell marker), CD10 (endometrial stromal cell marker) and by qPCR using PECAM1 (endothelial marker) and EPCAM (epithelial marker). This method produces 95–99% pure stromal cells.

Proestling K., Husslein H., Hudson Q.J., Witzmann-Stern M., Widmar B., Bagó-Horváth Z., Sandrieser L., Perricos A., Wenzl R, & Yotova I. (2023). MLLT11 Regulates Endometrial Stroma Cell Adhesion, Proliferation and Survival in Ectopic Lesions of Women with Advanced Endometriosis. International Journal of Molecular Sciences, 25(1), 439.

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Estrogen Detection Protocol for Cell and Animal Studies

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For E₂ detection, DMEM/F-12 without Phenol Red (Gibco, 11039021) and charcoal-stripped FBS (Biological Industries, 04–201-1A) were used. KGN cells were transfected with siRNA or pretreated with CQ or RAP for 48 h, while mouse GCs were pretreated with CQ for 48 h. The cells were then incubated in culture medium containing 10 nM testosterone (Sigma, T1500). After 24 h, the supernatant was collected for E₂ detection by electrochemistry, and all remaining cells were collected to extract total protein and to determine the concentration using the BCA kit. Finally, E₂ production was evaluated after being normalized to the total amount of protein. For the in vivo analysis, after injection of CQ for 5 days, mice were sacrificed and blood was collected. The blood samples were immediately placed at 4°C and incubated overnight and centrifuged at 1057 × g at 4°C to separate the supernatant. The E₂ concentration was measured by radioimmunoassay (Beijing North Institute of Biotechnology, China).

Shao T., Ke H., Liu R., Xu L., Han S., Zhang X., Dang Y., Jiao X., Li W., Chen Z.J., Qin Y, & Zhao S. (2022). Autophagy regulates differentiation of ovarian granulosa cells through degradation of WT1. Autophagy, 18(8), 1864-1878.

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Rat Cell Culture Reagents and Antibodies

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Sprague–Dawley rats were purchased from Charles River Japan (Hino, Japan). Dulbecco's modified Eagle's medium (DMEM) and DMEM/F-12 without phenol red, fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid (EDTA), and penicillin-streptomycin were purchased from GIBCO BRL (Grand Island, NY, USA). Human plasminogen and bovine fibrinogen were obtained from Enzyme Research Laboratories, Inc. (Swamsea, UK). Human urokinase and bovine thrombin were purchased from Calbiochem (Darmstadt, Germany). Monoclonal antibody for α-smooth muscle actin was purchased from DAKO (Glostrup, Denmark). 2′,7′-dichlorofluorescin diacetate (DCF-DA) was obtained from Molecular Probes (Eugene, OR, USA) and was dissolved in dimethyl sulfoxide (DMSO). 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP) and 4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]-pyrimidin-3-yl]phenol (PHTPP) were purchased from Tocris Bioscience (Bristol, UK). LysoPC and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). E₂ was dissolved in ethanol (EtOH), whereas ICI 182,780, MPP, and PHTPP were dissolved in DMSO. All of the other chemicals were dissolved in water.

Yoon B.K., Kang Y.H., Oh W.J., Lee D.Y., Kim D.K., Kessel B, & Kang C.D. (2020). Effects of 17β-Estradiol on the Plasminogen Activator System in Vascular Smooth Muscle Cells Treated with Lysophophatidylcholine. Journal of Menopausal Medicine, 26(1), 9-17.

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