Mcdb131 media

Manufactured by Thermo Fisher Scientific

Sourced in Singapore, United States

MCDB131 media is a basal medium designed for the culture of a variety of mammalian cell lines. It provides the basic nutrients and supplements required for cell growth and maintenance in vitro.

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MCDB131 (91 citations)

12 protocols using mcdb131 media

Isolation and Culture of Primary Human Cells

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Whole human blood was collected in heparinized vacutainers, and peripheral blood mononuclear cells (PBMCs) were collected and isolated in CPT Vacutainers™ (Becton Dickinson, Franklin Lakes, NJ) from healthy volunteers providing written informed consent in accordance with the University of Minnesota Institutional Review Board approved protocol (IRB Protocol 1506M74641). PBMCs were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM penicillin, and 50 µg/mL streptomycin (complete RPMI). Human foreskin fibroblasts (HFFs, ATCC catalog No. SCRC-1041) were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM penicillin, and 50 µg/mL streptomycin (complete DMEM). Human Skeletal Muscle cells (SkMC, Lonza, Basel, Switzerland, catalog No. CC-2561) were cultured in skeletal muscle cell culture media supplemented with a proprietary skeletal muscle cell media (BulletKit™, Lonza, Basel, Switzerland). HMEC-1 cells (dermal microvascular endothelial cells, ATCC catalog No. CRL3243) were cultured in MCDB131 media (Thermo Fisher, Waltham, MA) supplemented with 10 ng/mL epidermal growth factor, 1 µg/mL hydrocortisone, 10 mM glutamine, and 10% fetal bovine serum. Cells were cultured in 96-well plates at 37°C for 3.5–16 h, as indicated for that experiment.

Salyer A.C, & David S.A. (2018). Transcriptomal signatures of vaccine adjuvants and accessory immunostimulation of sentinel cells by toll-like receptor 2/6 agonists. Human Vaccines & Immunotherapeutics, 14(7), 1686-1696.

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Phytochemical Analysis of Okra

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Okra (mature and ready to be consumed) was purchased from local supermarkets in 2019 when the study is conducted. Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), ascorbic acid, Acarbose, HPLC grade water, sea sand and formic acid were purchased from Sigma-Aldrich (Singapore). All cell culture reagents, including trypsin, MCDB-131 media, Fetal Bovine Serum (FBS), Attachment Factor (AF), 0.4% trypan blue and L-glutamine acid were purchased from Thermofisher Scientific (Singapore). All other reagents used throughout the experiments in this study were of standard analytical grade.
The whole okra plants were washed and dried before they were separated into various parts, consisting of the Seeds, Outer Skin and Inner Skin. Subsequently, the various okra parts were oven-dried at 40 °C for 24 h [23 (link)]. The samples were crushed using mortar and pestle after being cooled down to generate a more homogeneous and representative sample. Samples were sieved individually to separate into following particle sizes: below 0.3 mm, between 0.3 mm and 2.8 mm, and above 2.8 mm. Fine samples with particle size below 0.3 mm was used for chemical analysis.

Ong E.S., Oh C.L., Tan J.C., Foo S.Y, & Leo C.H. (2021). Pressurized Hot Water Extraction of Okra Seeds Reveals Antioxidant, Antidiabetic and Vasoprotective Activities. Plants, 10(8), 1645.

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Culturing Human Lung Fibroblasts and Endothelial Cells

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Human lung CRL-1490 fibroblasts (ATCC; Manassas, VA) were maintained in Eagle’s Minimum Essential Medium (MEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 100 U/ml penicillin (Hyclone), 100 mg/ml streptomycin (Hyclone) and Amphotericin B (Invitrogen). Human umbilical vein endothelial cells (HUVECs) were cultured in MCDB131 media (Gibco) supplemented with 25% FBS, 0.05% brain bovine extract (BD Biosciences), 0.25% endothelial cell growth supplement (BD Biosciences), 0.1% heparin (Sigma), 1% L-glutamine (Sigma) and 0.1% gentamicin sulphate (Sigma). HUVECs between passages two and seven were used. Both CRL-1490 and HUVEC cell lines were cultured at 37°C in 5% CO₂ incubator, and were passaged at preconfluent densities using a solution containing 0.05% trypsin (Sigma) and 0.5 mM EDTA (Invitrogen).

Iyer A.K., Ramesh V., Castro C.A., Kaushik V., Kulkarni Y.M., Wright C.A., Venkatadri R., Rojanasakul Y, & Azad N. (2015). Nitric Oxide Mediates Bleomycin-Induced Angiogenesis and Pulmonary Fibrosis via Regulation of VEGF. Journal of cellular biochemistry, 116(11), 2484-2493.

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Culturing Human Microvascular Endothelial Cells

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Human Microvascular Endothelial cells (HMEC-1, #CRL-3243, ATCC, Manassas, VA) were maintained in complete growth media. Complete growth media consisted of MCDB 131 media (#10372019, Gibco, Grand Island, NY) supplemented with 2 mM L-glutamine (#25030081, Thermo Fisher Scientific), 10% (v/v) FBS (#15000044, Thermo Fisher Scientific) and penicillin/streptomycin (100 U/ml and 100 μg/ml concentration, respectively). Cells were seeded at a density of 10,000 cells/cm² onto fibronectin-coated glass coverslips (#CS-25R17, thickness 1.5, Warner Instruments, Hamden CT) (fibronectin, 20 μg/ml, #33016–015, Thermo Fisher Scientific) mounted in 6-well plates. The cells were grown to confluence in a humidified environment at 37°C with 5% CO₂.

Williamson L.M., Lowe S., Love E.M., Cohen H., Soldan K., McClelland D.B., Skacel P, & Barbara J.A. (1999). Serious hazards of transfusion (SHOT) initiative: analysis of the first two annual reports. BMJ : British Medical Journal, 319(7201), 16-19.

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Cell Line Culture and Validation

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SCCHN cell lines (T. Carey, University of Michigan) were cultured as described and validated by genotyping at the Sequencing Core (University of Michigan) before and at the end of the study (24 (link)–25 (link)). An immortalized human microvascular endothelial cell line HMEC-1 (Center for Disease Control) was used for sprouting assays. HMEC-1 cells were maintained in MCDB-131 media (Gibco) supplemented with 10% FBS, 1% glutamine, 1% penicillin/streptomycin and 1 ng/ml epidermal growth factor.

Banerjee R., Van Tubergen E.A., Scanlon C.S., Broek R.V., Lints J.P., Liu M., Russo N., Inglehart R.C., Wang Y., Polverini P.J., Kirkwood K.L, & D’Silva N.J. (2014). The G-protein Coupled Receptor GALR2 Promotes Angiogenesis in Head and Neck Cancer. Molecular cancer therapeutics, 13(5), 1323-1333.

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Isolation and Lysis of Mouse Brain Microvasculature

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Brain microvasculature was isolated from whole mouse brain as previously described⁸⁵. Briefly, brains were dissected, cut into small pieces, and homogenized in MCDB131 media (Gibco, Waltham, MA, USA) with 2% Cosmic calf serum (HyClone, Logan, UT, USA) with a dounce homogenizer. Homogenates were suspended in MCDB131 media with 17% dextran (70,000 MW, Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 10,000 RPM in a swing bucket rotor. The pellet was resuspended in 1X cell lysis buffer (Cell Signaling Technology, 9803, Danvers, MA, USA) with protease inhibitors (cOmplete mini, Roche, Indianapolis, IN, USA), briefly sonicated, and centrifuged at 12,000 × g at 4 °C for 15 min. Protein lysates were analyzed using either traditional Western blot or capillary electrophoresis immunoassays.

Hussong S.A., Banh A.Q., Van Skike C.E., Dorigatti A.O., Hernandez S.F., Hart M.J., Ferran B., Makhlouf H., Gaczynska M., Osmulski P.A., McAllen S.A., Dineley K.T., Ungvari Z., Perez V.I., Kayed R, & Galvan V. (2023). Soluble pathogenic tau enters brain vascular endothelial cells and drives cellular senescence and brain microvascular dysfunction in a mouse model of tauopathy. Nature Communications, 14, 2367.

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HMEC-1 Cell Culture Protocol

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Human microvascular endothelial cells (HMEC-1) obtained from the Center for Disease Control (Atlanta, Georgia), were cultured in MCDB-131 media (Gibco) supplemented with 10% fetal bovine serum (FBS), L- glutamine (2 mmol/L), human epidermal growth factor (10 ng/ml), penicillin/streptomycin (50 μg/ml) and hydrocortisone (1 μg/ml) in a CO 2 /O 2 incubator at 37 °C.

Khalil A., Poelvoorde P., Fayyad-Kazan M., Rousseau A., Nuyens V., Uzureau S., Biston P., El-Makhour Y., Badran B., Van Antwerpen P., Boudjeltia K.Z, & Vanhamme L. (2018). Apoliporotein L3 interferes with endothelial tube formation via regulation of ERK1/2, FAK and Akt signaling pathway. Atherosclerosis, 279.

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Lentivirus Production and Endothelial Cell Culture

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Low-passage (P16) 293T cells purchased from the American Type Culture Collection (ATCC, Manassas, VA) were used for the production of lentivirus particles. The MS1 (mouse pancreatic islet) endothelial cell line was also purchased from the ATCC. Immortalized H5V (mouse heart endothelial cells) were previously acquired (23 (link)). 293T, MS1, and H5V cell lines were grown in RPMI supplemented with 10% FBS, 100 IU mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin sulfate. HMEC-1 cells, an SV40 transformed human microvascular endothelial cell line (24 (link)), were obtained from the Centers for Disease Control and Prevention (Atlanta, GA) and grown in MCDB 131 media (Invitrogen, Carlsbad, CA) containing mouse EGF (BD, Franklin Lakes, NJ), hydrocortisone (Sigma-Aldrich, St. Louis, MO), 10% FBS, 100 IU mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin sulfate and 1x GlutaMax (Invitrogen, Carlsbad, CA). All cell lines used were tested by the University of Pennsylvania Cell Center (Philadelphia, PA) and found to be mycoplasma-free. No other authentication assay was performed.

Santoro S.P., Kim S., Motz G.T., Alatzoglou D., Li C., Irving M., Powell DJ J.r, & Coukos G. (2014). T cells bearing a chimeric antigen receptor against prostate-specific membrane antigen mediate vascular disruption and result in tumor regression. Cancer immunology research, 3(1), 68-84.

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Endothelial and Ovarian Cancer Cell Cultivation

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HMVEC cells (Lonza) were expanded and used from passage 3–7. Endothelial cells were grown using EGM-2MV media (Lonza) on cell culture plates coated with 0.2% gelatin. HMEC-1 cells, an SV40 transformed human microvascular endothelial cell line ^{46 (link)}, were obtained from the Centers for Disease Control and Prevention (Atlanta, GA) and grown in MCDB 131 media (Invitrogen) containing mouse EGF (BD), hydrocortisone (Sigma), 10% FBS, 1% PenStrep and 1× GlutaMax (Invitrogen). Ovarian cancer cell lines were obtained from either from ATCC or were a gift from the laboratory of Carl June developed from primary ovarian tumors. All lines were grown in RPMI containing 10% FBS and 1% PenStrep.

Motz G.T., Santoro S.P., Wang L.P., Garrabrant T., Lastra R.R., Hagemann I.S., Lal P., Feldman M.D., Benencia F, & Coukos G. (2014). Tumor Endothelium FasL Establishes a Selective Immune Barrier Promoting Tolerance in Tumors. Nature medicine, 20(6), 607-615.

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Endothelial and Ovarian Cancer Cell Cultivation

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