Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (San Diego, CA) and cultured in endothelial basal medium 2 (EBM‐2) plus 10% FBS and EGM‐2 MV SingleQuotes (Clonetics) in a humidified atmosphere of 5% CO2 and 95% air. HUVECs were seeded into 6‐well plates at 5×105 cells per well and cultured overnight in serum‐free EBM‐2. HUVECs at passage 5 to 8 were used. After washing twice with PBS, cells were treated with carbachol (Sigma‐Aldrich, St. Louis, MO) and/or donepezil (Selleck Chemicals, Houston, TX) at indicated concentrations and time points. In some experiments, HUVECs were treated with a phosphoinositide 3‐kinase (PI3K) inhibitor (LY294002; Cell Signaling Technology, Beverly, MA) at indicated concentrations. Lysates and conditioned media were then collected for biological analysis.
Huvecs
Manufactured by Cell Signaling Technology
Sourced in United States
HUVECs (Human Umbilical Vein Endothelial Cells) are primary cells derived from the human umbilical vein. They are used as an in vitro model to study endothelial cell biology and function.
Automatically generated - may contain errors
Lab products found in correlation
Donepezil, by Selleck Chemicals (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Huvecs, by Cambrex (1 mentions)
Carbachol, by Merck Group (1 mentions)
Egm 2 mv singlequotes, by Cambrex (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Huvecs, by Cell Applications (1 mentions)
Penicillin, by ScienCell (1 mentions)
Atg5 sirna, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
Huvecs, by ScienCell (1 mentions)
Endothelial cell growth supplement (ecgs), by ScienCell (1 mentions)
Huvecs, by R&D Systems (1 mentions)
Paricalcitol, by Bio-Techne (1 mentions)
Huvecs, by Bio-Techne (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Huvecs, by Lonza (1 mentions)
4 protocols using huvecs
1
HUVEC Culture and Carbachol/Donepezil Treatment
2
Tissue Protective Peptide Activates AKT Signaling
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Example 2
This example shows that a tissue protective peptide activates AKT.
HUVECs were purchased from Cell Applications (San Diego, Calif.) and grown in medium EGM-2 supplemented with 2% fetal calf serum (FCS) and penicillin/streptomycin (P/S) at 37° C. under air containing 5% CO2 in a humidified incubator. Preliminary experiments showed that phosphorylation of Akt reached a maximum 10 minutes following stimulation by compounds that activated the tissue protective receptor. After HUVECs were treated with GDKA (SEQ ID NO: 3) peptide, cell lysates were prepared and Western blot was performed using anti-Akt and anti-phospho-Akt antibodies (Cell Signaling Technology, Danvers, Mass.). Briefly, cell lysates were subjected to SDS-PAGE, and transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, N.J.). The membrane was blocked for 1 h at room temperature with PBS containing 2% BSA and 0.05% Tween 20. The blots were incubated overnight at 4° C. or for 4 h at room temperature with a primary antibody against phospho-Akt, followed by incubation for 1 h with a secondary, horseradish peroxidase-conjugated antibody. Then the blots were reprobed with an antibody against Akt to confirm equal protein loading. Immuno-reactive bands were visualized using ECL (Amersham Biosciences, Piscataway, N.J.).
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3
Endothelial Cell Autophagy Regulation
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HRGECs and HUVECs were obtained from ScienCell Research Laboratories (4000, 8000, ScienCell; Santiago, USA) and cultured with endothelial cell medium (1001, ScienCell; Santiago, USA), containing 10% FBS (0025, ScienCell; Santiago, USA), 5 ml endothelial cell growth supplement (1052, ScienCell; Santiago, USA) and 5 ml penicillin (10,000 U/ml)/streptomycin (10,000 µg/ml) solution (0503, ScienCell; Santiago, USA). Cells were incubated with IL-6 at indicated time points or different concentrations of IL-6 for 24hours. 3-MA (10 mM) was used as an autophagy inhibitor in vitro experiment.
For transfection, HUVECs that were cultured in 6-well plates with non-proliferative medium. The HUVECs were transfected with 25nM of ATG5 siRNA (6345, Cell Signaling Technology; Boston, USA) or control siRNA using Lipofectamine 2000 transfection reagent (11668–019, Invitrogen; California, USA) according to the manufacturer’s instructions.
For transfection, HUVECs that were cultured in 6-well plates with non-proliferative medium. The HUVECs were transfected with 25nM of ATG5 siRNA (6345, Cell Signaling Technology; Boston, USA) or control siRNA using Lipofectamine 2000 transfection reagent (11668–019, Invitrogen; California, USA) according to the manufacturer’s instructions.
4
Paricalcitol Attenuates TNF-α-Induced Endothelial Activation
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Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza Walkersville, Inc. (Walkersville, MD, USA) and cultured in endothelial basal medium (EBM)-2 complete medium supplemented with 2% (v/v) heat-inactivated fetal bovine serum at 37°C in 5% CO2. To examine the effects of paricalcitol on the LPS-induced increase in cell adhesion molecule expression in HUVECs, subconfluent endothelial cells were incubated with paricalcitol (Tocris Bioscience, Minneapolis, MN, USA; 0.01, 0.1 or 1 µM) for 30 min and then stimulated with 10 ng/ml TNF-α (R&D Systems, Minneapolis, MN, USA) for 6 h.
For p65 immunocytochemistry, the HUVECs were cultured in 6-well plates and treated with 1 µM paricalcitol for 30 min and then stimulated with 10 ng/ml TNF-α for 6 h. Following 4% paraformaldehyde fixation, the cells were washed with phosphate-buffered saline (PBS), permeabilized in 0.25% Triton X-100 and blocked with 1% goat serum. The HUVECs were then incubated with primary anti-p65 antibody (#4764; Cell Signaling Technology, Beverly, MA, USA), followed by staining with Cy3-conjugated secondary antibody (AP182C; Chemicon, Temecula, CA, USA). For nuclear staining, the HUVECs were incubated with 4′,6-diamidino-2-phenylinole (DAPI; Molecular Probes, Eugene, OR, USA).
For p65 immunocytochemistry, the HUVECs were cultured in 6-well plates and treated with 1 µM paricalcitol for 30 min and then stimulated with 10 ng/ml TNF-α for 6 h. Following 4% paraformaldehyde fixation, the cells were washed with phosphate-buffered saline (PBS), permeabilized in 0.25% Triton X-100 and blocked with 1% goat serum. The HUVECs were then incubated with primary anti-p65 antibody (#4764; Cell Signaling Technology, Beverly, MA, USA), followed by staining with Cy3-conjugated secondary antibody (AP182C; Chemicon, Temecula, CA, USA). For nuclear staining, the HUVECs were incubated with 4′,6-diamidino-2-phenylinole (DAPI; Molecular Probes, Eugene, OR, USA).
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