Choroidal or retinal endothelial cells were obtained from non-diseased eye pairs of 8 human cadaveric donors (Lions VisionGift), adhering closely to our previously published method.16 (link) In brief, choroid or retina were dissected from posterior eye cups and digested with graded solutions of Dispase (Life Technologies-Gibco, Carlsbad, CA) and type II collagenase (Sigma-Aldrich, St. Louis, MO) in MCDB-131 medium (Sigma-Aldrich) with 2% FBS. Endothelial cells were isolated from the digested tissue using magnetic Dynabeads (Dynal-Invitrogen, Oslo, Norway) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA), per the manufacturer’s instructions. Cells were cultured at 37 °C and 3.5% to 5% CO2 in MCDB-131 medium (Sigma-Aldrich) with up to 10% FBS and EGM-2 SingleQuots supplement, omitting gentamicin, hydrocortisone and FBS (Lonza-Clonetics, Walkerville, MD). To generate sufficient cells for study, the cells were transduced with the mouse recombinant amphotropic retrovirus, LXSN16E6E7 (gift of Denise A. Galloway, PhD, Fred Hutchinson Cancer Institute).
Mcdb 131 medium
Manufactured by Merck Group
Sourced in United States, Italy
MCDB 131 medium is a cell culture medium designed for the growth and maintenance of a variety of cell lines. It provides the necessary nutrients and components to support cell proliferation and survival in an in vitro environment.
Automatically generated - may contain errors
Lab products found in correlation
Hydrocortisone, by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
G5 supplement, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Hmec 1, by American Type Culture Collection (3 mentions)
Hydrocortisone, by Lonza (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Epidermal growth factor (egf), by Merck Group (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Biowest (2 mentions)
Streptomycin, by Biowest (2 mentions)
Epidermal growth factor (egf), by ProSpec (2 mentions)
Collagenase type 2, by Merck Group (2 mentions)
Gentamicin, by Lonza (2 mentions)
Egm 2 singlequot, by Lonza (2 mentions)
Egm 2 singlequots supplement, by Lonza (2 mentions)
Recombinant human fibroblast growth factor basic rhfgf 2, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
33 protocols using mcdb 131 medium
1
Isolation and Culture of Human Choroidal and Retinal Endothelial Cells
2
Glioblastoma Cell Culture Protocol
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GBM cell lines, primary GBM cells, and normal human astrocytes were cultured as described previously (7 (link), 14 (link), 47 (link)). In brief, GBM cell lines A172, LN-18, SF-268, SF-295, T98MG, U251, and U87MG were maintained in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% EquaFETAL® bovine serum (Atlas Biologicals, Inc.) and 100 μg/ml streptomycin and 100 IU/ml penicillin (Gibco). Primary cells VTC-001, VTC-002, VTC-004, VTC-037, VTC-056, VTC-058, VTC-084, and VTC-103 were cultured in DMEM supplemented with 15% fetal bovine serum (Peak Serum, Inc.) and penicillin/streptomycin. Normal human astrocytes were cultured in MCDB-131 Medium (Sigma) containing 3% fetal bovine serum (Peak Serum, Inc.), 10 X G-5 Supplement (Gibco), and penicillin/streptomycin. Cell lines have been authenticated by the ATCC authentication service utilizing Short Tandem Repeat (STR) profiling. Primary GBM cells were kept at low passages (no more than 10). Antibodies of RAN and GAPDH were purchased from Santa Cruz Biotechnology, Inc. Importazole was purchased from Cayman Chemicals, Inc. Stock solution of importazole was prepared at 50 mM using dimethyl sulfoxide (DMSO). Working solution was further diluted using cell culture media.
3
Microvascular Endothelial Cell Inflammation
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Human microvascular endothelial cells (HMEC-1) were gifted from Dr. Moonsoo M Jin (Weill Cornell Medicine). HMEC-1 cells were cultured in MCDB 131 medium (Sigma) supplemented with 10% FBS (Natocor—Industria Biológica, Argentina), 1 mg/ml hydrocortisone (Sangon Biotech, Shaihai, China), and 10 ng/ml recombinant human epidermal growth factor (Sangon Biotech, Shaihai, China). For induction of inflammation, HMEC-1 cells were treated with 1 mg/ml of LPS 3 h after the treatment with herbal extracts. The mammalian cells were maintained at 37°C in a 5% CO2 humidity incubator.
4
Angiogenesis Regulation by IGFBP7 and VEGFA
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MCDB131 medium, endothelial cell growth supplement (ECGS), and CoCl2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human IGFBP7 and VEGFA were obtained from R&D Systems (Minneapolis, MN, USA). Ovine LH (NIDDK, oLH26, AFP-5551B) was provided by Prof AF Parlow of the National Hormone and Pituitary Program, Harbor/UCLA Medical Center. Growth factor-reduced Matrigel (BD Biosciences, MA, USA) was gelatinated at 37 C in a CO2 incubator for 30 min prior to the Matrigel assay. WST-8 reagent (Cell Counting Kit, Dojindo, Tokyo, Japan) was used to evaluate the proliferation of LECs. Poly(A)+ RNA was isolated using a QuickPrep Micro mRNA Purification Kit (GE Healthcare, Buckinghamshire, UK). The Phospho- ERK1/2 (phospho-extracellular signal-regulated kinase 1/2) Pathway Kit and an MEK1/2 (anti-mitogen-activated protein kinase kinase 1/2 antibody) were purchased from Cell Signaling Technology (Beverly, MA, USA).
5
Culturing HMEC-1 Cells for Experiments
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The HMEC-1 cell line (Centers for Disease Control and Prevention, Atlanta, GA, USA) was kindly gifted by Prof. Nicoletta Basilico, Dipartimento di Scienze Biomediche, Chirurgiche e Odontoiatriche, Università degli Studi di Milano (Italy). Cells were grown in MCDB 131 medium (Sigma Aldrich) plus 1% penicillin/streptomycin, 10% fetal bovine serum (FBS), 20 mM HEPES buffer, 1 μg/mL hydrocortisone and 10 ng/mL epidermal growth factor (EGF) [31 (link)]. Cells were maintained at 37 °C in a 5% CO2 atmosphere and passaged every 3/4 days. For experiments, where not differently stated, cells’ growth lasted 48 h.
6
Propagation and Maintenance of Endothelial Cell Lines
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The human pulmonary microvascular endothelial cell line HPMEC-ST1.6R was kindly donated by Dr. J.C. Kirkpatrick (Institute of Pathology, Johannes Gutenberg University, Germany) and propagated (passages 5–8) and maintained at 37°C in humidified air with 5% CO2 in endothelial cell basal medium-2 supplemented with growth factors, antibiotics, and fetal bovine serum as per the manufacturer’s specifications (Clonetics, Lonza). The human dermal microvascular endothelial cell line HMEC-1 was kindly donated by Dr. M. Welch (University of California, Berkeley) and propagated (passages 20–25) and maintained at 37°C in humidified air with 5% CO2 in MCDB 131 medium (Sigma) supplemented with 0.2% Epidermal Growth Factor and 0.4% hydrocortisone. Human Umbilical Vein microvascular endothelial cells (HUVEC) were grown as previously described [9 (link)].
7
Culturing Human Microvascular Endothelial Cells
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Human dermal microvascular endothelial cell 1 (HMEC-1) was cultured (37 °C, 5% CO2) in MCDB131 medium (Sigma-Aldrich, St. Louis, MI, USA), supplemented with hydrocortisone (1 μg/mL; Sigma-Aldrich, St. Louis, MI, USA), recombinant human endothelial growth factor (EGF; 1 ng/mL; Gibco, USA), fetal bovine serum (FBS; 10% (v/v), Gibco, USA), and penicillin-streptomycin (100 U/mL) (Lubrano et al., 2012), as described [19 (link)]. Experiments were performed in a medium without FBS (minimum medium).
8
Culture and Maintenance of Human Umbilical Vein Endothelial Cells
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Human umbilical vein endothelial cells (HUVECs) were purchased from Kurabo (Osaka, Japan) and stored in liquid nitrogen. The cells were maintained on type-I collagen-coated dishes in MCDB131 medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; JRH Bioscience, Lenexa, KS), 10 ng/mL EGF, 5 ng/mL bFGF, 50 μg/mL heparin, 100 U/mL penicillin G, and 100 μg/mL streptomycin sulfate at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. HUVECs at the third to fifth passages were used in experiments. In some experiments, the medium from which FCS had been deleted was used as serum-free basal medium for HUVECs.
9
Culturing HMEC-1 and THP-1 cells
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Human micro-vascular endothelial cell-1 (HMEC-1, from American Type Culture Collection Manassas, USA) was maintained in MCDB 131 medium (Sigma, USA) with L-glutamine and fetal bovine serum (Sigma, USA) to a final concentration of 15%. The medium was supplemented with 500 units/ml penicillin (Biowest, USA), 500 μg/ml streptomycin (Biowest, USA), 10 ng/ml Epidermal Growth Factor (ProSpec, USA) and 1□g/ml hydrocortisone (Sigma, USA). The THP-1 cells were maintained in the RPMI-1640 medium (Invitrogen, USA) with 10% FBS.
10
Establishment of Primary Glioblastoma Cell Line
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Commercially available human GBM cell lines, including U87 MG (ATCC® HTB-14™) and A172 (ATCC® CRL-1620™), were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). Normal human astrocytes (NHA) and human GBM cell line U251 were both obtained from the China Academia Sinica Cell Repository (Shanghai, China). GBM cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco), while NHA cells were grown in MCDB-131 medium (Sigma; St. Louis, MO, USA) containing 3% FBS and 10× G-5 Supplement (Gibco). Primary glioblastoma cell line was established as previously described [24 (link)]. Briefly, tumor tissue was obtained from one GBM patient. After removing the vessels, clotted blood, and charred tissue, sample was dissociated by Collagenase Type IVa (250 U/mL) and Pronase E (2.5 U/mL) for 1 h at 37°C. Then, cells were centrifuged at 300 × g for 5 min at 4°C, resuspended in DMEM containing 10% FBS, and put in a cell culture flask. The cell culture medium was changed every 2 days. Cells were all maintained in a humidified incubator with an atmosphere of 5% CO2 at 37°C.
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