Mcdb131

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany, Spain

MCDB131 is a basal medium formulation designed for the in vitro cultivation of a variety of mammalian cell types. It contains a balanced salt solution, amino acids, vitamins, and other components required for cell growth and maintenance. The medium is suitable for use in a range of cell culture applications, including research and biopharmaceutical production.

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Complete MCDB131 medium (5 citations) MCDB131 media (12 citations)

91 protocols using mcdb131

Cell Culture Protocols for Cancer and Endothelial Cells

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LNCaP (ATCC, CRL—1740), Du-145 (ATCC, CRL—HTB—81), PC3 (ATCC, CRL—1435), C4-2 (ATCC, CRL—3314), HEK293E (ATCC, CRL—10852) and Jurkat cells (ATCC, TIB-152) were cultured in RPMI medium 1640 (Gibco, Waltham, Massachusetts, USA #61870-010) supplemented with 10% foetal bovine serum (FBS) (Gibco, #10500-064) and 1% penicillin–streptomycin (Gibco, #15140-122). Human dermal microvascular endothelial cells (HMEC-1) stably overexpressing human FcRn (HMEC-1-FcRn) [18 (link)] were cultivated in complete medium consisting of MCDB131 (Life Technologies, Waltham, Massachusetts, USA #10372-019), Recombinant human EGF (PeproTech, Cranbury, New Jersey, USA #AF-100-15), Hydrocortisone (Sigma-Aldrich, St. Louis, Missouri, USA #H0888), G418 solution (Sigma-Aldrich, #G418-RO), Puromycin (Life Technologies, #A11138-03), 10% FBS, and 2 mM l-glutamine (Lonza, Basel, Switzerland #BE17-605E). Cells were cultivated following ATCC protocols, and mycoplasma tested when new stock were thawed. Cell lines were routinely authenticated by short tandem profiling.

Glud E.N., Rasmussen M., Zhang Y., Mandrup O.A., Salachan P.V., Borre M., Sørensen K.D, & Howard K.A. (2022). Identification of a high-risk immunogenic prostate cancer patient subset as candidates for T-cell engager immunotherapy and the introduction of a novel albumin-fused anti-CD3 × anti-PSMA bispecific design. British Journal of Cancer, 127(12), 2186-2197.

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Generating CAF-like Cells from HMEC-1

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CAF-like cells were obtained by TGF-β2 stimulation of human microvascular endothelial cells (HMEC-1) as described previously [35 (link),36 (link),37 (link)]. HMEC-1 were cultured in MCDB131 (Life Technologies, Paisley, UK) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies) or with one of CM: co-CM1, co-CM2, co-CM1 + VIN, co-CM2 + VIN, CAFs-CM1, CAFs-CM2, CAFs-CM1 + VIN, CAFs-CM2 + VIN, or CAFs-CM. All lines were maintained at 37 °C in a humidified 5% CO₂ atmosphere. CM was recovered from colon cancer cells grown for 72 h. In some experiments, the cells were treated with vincristine (5 nM) or NSAIDs: 2500 µM aspirin (AsA) and 400 µM ibuprofen (IBU). The cells were harvested by 0.05% trypsin-EDTA and washed with phosphate-buffered saline (PBS).

Wawro M.E., Sobierajska K., Ciszewski W.M, & Niewiarowska J. (2019). Nonsteroidal Anti-Inflammatory Drugs Prevent Vincristine-Dependent Cancer-Associated Fibroblasts Formation. International Journal of Molecular Sciences, 20(8), 1941.

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Ovarian Cancer Cell Lines Cultivation

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The human ovarian HEY cell line was derived from a human ovarian cancer xenograft originally grown from a peritoneal deposit of a patient with moderately differentiated papillary cystadenocarcinoma of the ovary [54 (link)]. The human ovarian clear cell adenocarcinoma cell line TOV21G was derived from grade 3, stage III, primary malignant adenocarcinoma from a patient who was not exposed to chemotherapy or radiation therapy [55 (link)]. The HEY cell line was grown in complete RPMI 1640 (Sigma-Aldrich, Sydney, Australia) supplemented with 10% (v/v) heat inactivated foetal bovine serum (FBS) (Thermo Fisher Scientific, MA, USA), 2mM L-glutamine (Murdoch Childrens Research Institute, Victoria, Australia) and 1% (v/v) penicillin and streptomycin. The TOV21G cell line was maintained in 1:1 mixture of MCDB131 (Life Technologies, CA, USA) and Medium 199 (Lonza, Basel, Switzerland) supplemented with 15% (v/v) heat inactivated FBS (Thermo Fisher Scientific), 2mM L-glutamine and 1% (v/v) penicillin and streptomycin. Cells were routinely checked for mycoplasma infection.

Chan E., Luwor R., Burns C., Kannourakis G., Findlay J.K, & Ahmed N. (2018). Momelotinib decreased cancer stem cell associated tumor burden and prolonged disease-free remission period in a mouse model of human ovarian cancer. Oncotarget, 9(24), 16599-16618.

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Isolation and Characterization of Ascites Tumor Cells

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Ascites samples were processed within 24 hours of collection. Cells in the ascites were separated by the method developed in our laboratory as described previously (37 (link)). Briefly, approximately 100-500mL of ascites was centrifuged and the supernatant collected and stored at -80°C. Red blood cells from the cell pellet were removed by hypotonic shock. The remaining cells were suspended in a 1:1 ratio of MCDB131 (Life Technologies, CA, USA) and DMEM (Sigma-Aldrich), supplemented with 10% (v/v) heat inactivated FBS (Thermo Fisher Scientific, MA, USA), 2mM L-glutamine (Invitrogen Corporation, CA, USA), and an antibiotic combination of 1% (v/v) streptomycin and penicillin (Invitrogen Technologies, Vic, Australia). Approximately 1x10⁵-1x10⁶ tumor cells per well were seeded onto 6-well Corning^® Ultra-Low attachment plates (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37°C in 5% CO₂. Seeded cells grew into two distinct populations: the non-adherent epithelial cells and adherent mesenchymal population were collected and frozen in TRIzole (ThermoFisher Scientific, Melbourne, Australia) for RNA extraction.

Escalona R.M., Kannourakis G., Findlay J.K, & Ahmed N. (2022). Expression of TIMPs and MMPs in Ovarian Tumors, Ascites, Ascites-Derived Cells, and Cancer Cell Lines: Characteristic Modulatory Response Before and After Chemotherapy Treatment. Frontiers in Oncology, 11, 796588.

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Cytokine and Chemical Effects on Cells

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To determine the effect of cytokine and chemical exposure on gene expression, coagulation factor production, and surface FX activation, cells were washed with PBS before 24-hr exposure to either serum-free media alone (MCDB-131 + insulin-transferrin-selenium, Life Technologies), 10 ng/ml TNF, or 30 µg/ml ATA in serum-free media.

Cohen C.T., Turner N.A, & Moake J.L. (2020). Production and control of coagulation proteins for factor X activation in human endothelial cells and fibroblasts. Scientific Reports, 10, 2005.

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VEGF-A Stimulation of HUVECs: Immunoblot and Immunofluorescence Analysis

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HUVECs were serum starved in MCDB131 (Life Technologies) for 2 h prior to stimulation with 25 ng/mL VEGF‐A_165a for various times and lysed for immunoblotting or immunoprecipitation analyses. In some experiments, cells were pre‐treated with 10 µg/mL CHX (2 h) prior to VEGF‐A stimulation or with 20 µg/mL CHX at the same time as VEGF‐A stimulation. Lysate preparation and immunoblot analysis were performed as previously described 30, 31. For immunofluorescence analysis, cells were serum starved and pre‐treated with 10 µg/mL CHX for 2 h prior to stimulation with VEGF‐A before being fixed and processed as previously described 30, 31. Images were acquired using an Evos‐fI inverted digital microscope (Life Technologies) or a wide‐field deconvolution microscope DeltaVision (Applied Precision Inc.). Relative co‐distribution was quantified using NIH Image J (http://rsb.info.nih.gov/ij/).

Smith G.A., Fearnley G.W., Abdul‐Zani I., Wheatcroft S.B., Tomlinson D.C., Harrison M.A, & Ponnambalam S. (2015). VEGFR2 Trafficking, Signaling and Proteolysis is Regulated by the Ubiquitin Isopeptidase USP8. Traffic (Copenhagen, Denmark), 17(1), 53-65.

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Aortic Microvessel Sprouting Assay

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The assay was performed as previously described (58 (link)). The thoracic aorta was dissected from euthanatized 8-week-old SD rats and followed by transverse section into the ring shape. The aorta rings were embedded in the 1 ml mixtures of Matrigel and MCDB131 media (Life technologies Ltd.) (1:1, v/v). To investigate the effect of LECT2 on microvessel sprouting, the aorta rings were incubated with rLECT2-containing medium for 7 days. The length of microvessel sprouts was measured with the microscope and digital image system (Leica). Five fields in each aortic ring were randomly selected for quantification.

Chu T.H., Ko C.Y., Tai P.H., Chang Y.C., Huang C.C., Wu T.Y., Chan H.H., Wu P.H., Weng C.H., Lin Y.W., Kung M.L., Fang C.C., Wu J.C., Wen Z.H., Lee Y.K., Hu T.H, & Tai M.H. (2022). Leukocyte cell-derived chemotaxin 2 regulates epithelial-mesenchymal transition and cancer stemness in hepatocellular carcinoma. The Journal of Biological Chemistry, 298(10), 102442.

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Characterization of Thyroid Cancer Cell Lines

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Spontaneously immortalized human thyroid cancer cells: 8505c human ATC cells harboring the hemi/homozygous BRAF^V600E mutation were purchased from DSMZ (German collection of microorganisms and cell culture, Braunschweig, Germany)[9 (link); 27 (link)]. Human ATC SW1736 cells were provided by Dr. Nils-Erik Heldin (Uppsala University, Uppsala, Sweden), which harbor the heterozygous BRAF^WT/V600E mutation. Human thyroid cancer cells were grown in DMEM high glucose (CellGro, USA) medium supplemented with 10% fetal bovine serum (FBS) (CellGro, USA) and ampicillin/streptomycin. Primary human microvascular endothelial cells (blood vessel endothelial cells (BVECs) and lymphatic vessel endothelial cells (LVECs)) [28 ] were kindly provided from Dr. Harold F. Dvorak (BIDMC, Harvard Medical School, Boston, USA). BVECs and LVECs were grown in MCDB 131 (Life Technologies, USA) growth medium with additional glutgro (Corning, final concentration 2 mM) and MVGS (microvascular growth supplement) (Life Technologies, USA). For starvation conditions, MCDB 131 was supplemented with glutgro with 1% MVGS. All in vitro angiogenic and ELISA assays were performed on ATC cells cultured with the specific growth medium supplemented with no FBS.

Husain A., Hu N., Sadow P.M, & Nucera C. (2015). Expression of angiogenic switch, cachexia and inflammation factors at the crossroad in undifferentiated thyroid carcinoma with BRAFV600E. Cancer letters, 380(2), 577-585.

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Rat Aorta Angiogenesis Assay

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The microvessel sprouting in rat aortic rings was performed as previously described [21 (link)]. Briefly, thoracic aorta was dissected from euthanatized 8-week-old Sprague–Dawley rats and followed by transverse section into the ring shape. Each aorta ring was embedded in the 1 mL mixtures of Matrigel and MCDB131 media (Life technologies Ltd., Paisley, Scotland, UK; 1:1, v/v). For assessment of vascular sprouting, each well was further added 1 mL MCDB131 medium containing 10 nM α-MSH or/combination with 5 mM l-arginine onto the Matrigel and incubated in a 37 °C, 5% CO₂ chamber for 7 days. The length of vascular sprouts, the length from the aortic ring to the end of the greatest sprout was measured with the microscope and digital image system. Five fields in each aortic ring; 6 aortic rings in each group were randomly selected for quantification.

Weng W.T., Wu C.S., Wang F.S., Wu C.Y., Ma Y.L., Chan H.H., Wu D.C., Wu J.C., Chu T.H., Huang S.C, & Tai M.H. (2018). α-Melanocyte-Stimulating Hormone Attenuates Neovascularization by Inducing Nitric Oxide Deficiency via MC-Rs/PKA/NF-κB Signaling. International Journal of Molecular Sciences, 19(12), 3823.

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Partial Activation of HuMEC-1 Cells

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As previously described37 (link), HuMEC-1 were partially activated with 100 μg/ml LPS (Sigma Chemical Co. St. Louis, USA) in MCDB-131 (Life Technologies, Darmstadt, Germany), whereas the other fraction of HuMEC-1 remained non-activated. The treatment of every study group was performed for 60 min under different temperature conditions (Fig. 1A–C). After treatment, medium was removed and transmigration-assay was started.

Bogert N.V., Werner I., Kornberger A., Meybohm P., Moritz A., Keller T., Stock U.A, & Beiras-Fernandez A. (2016). Influence of hypothermia and subsequent rewarming upon leukocyte-endothelial interactions and expression of Junctional-Adhesion-Molecules A and B. Scientific Reports, 6, 21996.

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