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17 protocols using HUVECs

1

Expansion and Characterization of MSOD-B and HUVEC Cells

MSOD-B cells [20 ] were expanded in complete α-Minimum Essential Medium (αMEM) (CM) supplemented with 10% fetal bovine serum (FBS), 1% HEPES, 1% sodium pyruvate and 1% of penicillin–streptomycin–glutamine (PSG) solution (all from Gibco) and 5 ng/mL fibroblast growth factor-2 (FGF-2, R&D Systems) in a humidified incubator at 37 °C and 5% CO2. RFP + Human Umbilical Vein Endothelial cells (HUVECs) were purchased from Angio-Proteomie (cat# cAP-0001RFP) and expanded in 2D monolayers in Endothelial Growth Medium 2 (EGM-2) (Lonza; cat# CC-3162). At 90% confluency, cells were detached using Trypsin-EDTA 0.05% (Gibco), counted and seeded again at a density of 6000 cells per cm2.
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Human umbilical vein endothelial cells (HUVECs, Angio-Proteomie, Boston, MA, USA) were cultured in EGM-2 supplemented with BulletKit and 1% P/S. MDA-MB-231 and MCF7 breast cancer cells, and NIH/3T3 mouse fibroblasts (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM supplemented with 10% FBS and 1% P/S. Medium was changed every other day. The culture environment was maintained in a 37°C incubator with 5% humidified atmosphere of CO2.
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Human umbilical vein endothelial cells transfected with GFP-Lentiviral particles (HUVECs) were obtained from Angio-Proteomie (cat # cAP-0001GFP, Boston, MA). Cells were cultured in endothelial cell growth medium (EGM, cat # cAP-02, Angio-Proteomie) with 5% fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (PS). HUVECs from passage 4–6 were used. Primary cultures of bone marrow human mesenchymal stem cells (hMSCs) (donated by Dr. Brian Johnstone, OHSU Orthopedics) were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Corning Life Sciences, Fisher Scientific Co, Pittsburgh, PA) with 10% FBS and 1% penicillin/streptomycin. hMSCs were used from passage 2–4. All cells were maintained in a humidified incubator (5% CO2, 37 °C), and the culture media was changed every two days with one cell passage per week.
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NIH/3T3 cells (ATCC) were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% Glutamine and 1% penicillin/streptomycin (Biological Industries). Primary human umbilical vein endothelial cells (HUVECs, Angio-Proteomie) were maintained in EGM-2 (Lonza, Basel, Switzerland) supplemented as according to the manufacturer instructions. Neonatal cardiac cells were isolated according to Tel Aviv University ethical use protocols from intact ventricles of 1- to 3-day-old neonatal Sprague-Dawley rats. Cells were isolated using 6 cycles (37 °C, 30 min each) of enzyme digestion with collagenase type II (95 U mL-1, Worthington, Lakewood, New Jersey) and pancreatin (0.6 mg mL-1, Sigma-Aldrich) in DMEM. After each round of digestion, cells were centrifuged (600 g, 5 min) and resuspended in M-199 culture medium (Biological Industries) supplemented with 0.6 × 10-3 M CuSO4 · 5H2O, 0.5 × 10-3 M ZnSO4 · 7H2O, 1.5 × 10-3 M vitamin B12 (Sigma-Aldrich), 500 U mL-1 penicillin, 100 mg mL-1 streptomycin and 0.5% FBS. After the isolation procedure, cells were cultured in M-199 medium with 5% FBS and supplements as indicated above. Cell number was determined by a hemocytometer and trypan blue exclusion assay. Cells were cultured under a humidified atmosphere at 37°C with 5% CO2.
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Red fluorescent proteins (RFP) expressing human umbilical vein endothelial cells (HUVECs) were obtained from Angio-Proteomie (Boston, USA) and cultured in endothelial growth medium (EGM-2) and passage 5 was used for the experiments. 50 μl of precooled Matrigel (BD, Corning, USA) was coated into each well of a 96-well plate and polymerized for 30 min at 37 °C. Next, 2 × 104 HUVEC-RFP cells were suspended in a mixture of conditioned medium (50 μl) and endothelial cell medium (50 μl) containing 10% FBS. The fluorescence detection of tube formation was photographed using inverted microscopy (IX-53, Olympus) after 6 h of incubation at 37 °C.
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In our study, three different cell types were used to fulfill the experimental requirements, human umbilical vein endothelial cells (HUVECs; green fluorescent protein (GFP)-tagged or unlabeled; Angio-Proteomie, USA), MCF-7 breast tumor cells (ATCC, USA), and MDA-MB-231 breast cancer cells (HTB-26, ATCC). Endothelial growth medium (EGM, Lonza) supplemented with 1 v/v% antibioticantimycotic (ThermoFisher), was used to culture the HUVECs. Dulbecco’s modified Eagle’s medium (ThermoFisher) supplemented with 10 v/v% fetal bovine serum (ThermoFisher) and 1 v/v% antibioticantimycotic was used to culture MCF-7 cells or MDA-MB-231 cells. The cells were cultivated at 37 °C and 5 v/v% CO2 until 70% confluency. Prior to cell seeding, the expanded microchannel-embedded paper devices were autoclaved for 15 min to sterilize with simultaneous hydration. Subsequently, fibronectin (50 ng ml−1) was injected into the microchannels and incubated at 4 °C for 12 h for coating. HUVECs were seeded into the microchannels at a density of 1 × 107 cells ml−1 for 1 h, and the constructs were flipped for another hour to ensure uniform cell attachment on the microchannel surfaces. In the case of co-culture, after 12 h of HUVECs seeding, MCF-7 cells (2 × 106 cells ml−1) were further inoculated into the matrices of the paper devices and cultured in EGM.
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HUVECs purchased from Angio-Proteomie (Boston, MA, USA) were cultured in EGM-2 while human monocytes (THP-1) obtained from ATCC (Manassas, VA, USA) were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. The cell cultures were maintained at 37 °C and 5% CO2 in a standard incubator. The medium was replaced every 2–3 d and the cells cultured in flasks were subcultured when they reached approximately 80% confluency. For on-chip experiments, a 1:1 volume mixture of the two media was used as the common medium, where no adverse effects were observed on either cell type.
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The MM cell lines, MM.1S and H929, were purchased from the American Type Culture Collection (ATCC, Rockville, MD), OPM-2 and green fluorescent protein-labeled and luciferase-transfected MM.1S (MM.1S-GFP-Luc) were a kind gift from Dr. Irene Ghobrial (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA). Human umbilical vein endothelial cells (HUVECs) were purchased from Angio-Proteomie (Boston, MA). Human samples for this study were collected under informed consent, in concordance with Washington University Institutional Review Board (IRB) approval (IRB protocol number 201102270). MM cell lines were cultured in RPMI-1640 media (Corning, Tewksbury, MA) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies, Grand Island, NY), 2 mmol/L L-glutamine, 100 μg/mL penicillin, and 100 μg/mL streptomycin (Corning CellGro). HUVECs were cultured in Endothelial Growth Medium (EGM, Angio-Proteomie, Boston, MA) supplemented with endothelial growth supplements (including 10% FBS, recombinant growth factors, and 1% penicillin and streptomycin). All cells were cultured at 37 °C and in 5% CO2 in a NuAire water jacket incubator (Plymouth, MN).
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Human umbilical vein endothelial cells (HUVECs, Lonza) were cultured in endothelial growth medium 2 (EGM-2, Lonza) and passages 3–5 were used for the experiments. Endometrial epithelial cells (EECs: Ishikawa) and endometrial stromal fibroblasts (ESFs: CRL-4003 cells), obtained from ATCC and the laboratory of Dr Haeng Seok Song, respectively, were maintained in DMEM/F12 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin (Gibco, Grand Island, NY, USA) as previously described (Kang et al., 2015 (link)), and between passages 7 and 8 were used for all experiments. Red fluorescent protein (RFP)-labelled HUVECs (Angio-Proteomie; Boston, MA, USA) were procured at P3 and sub-cultured to produce P6 frozen stock. All cells were maintained in a humidified incubator at 37°C with 5% CO2.
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GFP- or RFP-expressing Human Umbilical Vein Endothelial Cells (HUVECs) (passage 4–6, Angioproteomie) were cultured on tissue culture flasks in EGM-2 medium supplemented with FBS (5% total FBS). Neonatal Human Dermal Fibroblasts (HNDFs) (passage 4–6, Clonetics) were cultured on tissue culture flasks in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% FBS (Hyclone), 1% non-essential amino acids (NEAA), 1% pen-strep and 0.2% b-mercaptoethanol (Sigma Aldrich).
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