Human umbilical vein endothelial cells (HUVEC) are a pool of endothelial primary cells derived from the human umbilical vein (Clonetics, Lonza, Switzerland). The HUVECs were cultured in an endothelial growth medium (EGM-2, Lonza, Switzerland), composed of endothelial basal medium (EBM-2, Lonza, Switzerland) and the SingleQuot Bullet Kit (Lonza, Switzerland). The HUVECs were seeded at a density of 5000/cm2 in T75 flasks (Corning Costar, Sigma Aldrich, St. Louis, MO, USA) and cultured at 37 °C and 5% CO2 with a daily change of the culture medium.
Singlequot bullet kit
Manufactured by Lonza
Sourced in Switzerland
The SingleQuot Bullet Kit is a laboratory equipment product designed for the efficient preparation of cell culture media. It provides a convenient and accurate way to supplement cell culture media with various growth factors, supplements, and other components required for cell cultivation. The kit contains pre-measured, sterile-filtered SingleQuot components that can be easily added to cell culture media, ensuring consistent and reliable media preparation.
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Lab products found in correlation
Ebm 2, by Lonza (4 mentions)
Egm 2, by Lonza (2 mentions)
Penicillin streptomycin, by Euroclone (1 mentions)
Rpmi 1640 medium, by Euroclone (1 mentions)
Thp 1 cell, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
Huvecs, by Cambrex (1 mentions)
T75 flasks, by Merck Group (1 mentions)
L glutamine, by Euroclone (1 mentions)
Cc 3132, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Human umbilical vein endothelial cells (huvec), by Keygen Biotech (1 mentions)
Ebm 2 medium, by Lonza (1 mentions)
Cc 2519, by Lonza (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Amphotericin, by Thermo Fisher Scientific (1 mentions)
Fungizone antimycotic, by Thermo Fisher Scientific (1 mentions)
Bronchial epithelial growth medium (begm), by Lonza (1 mentions)
8 protocols using singlequot bullet kit
1
Culturing Human Umbilical Vein Endothelial Cells
2
Culturing Human Umbilical Vein Endothelial Cells
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Human umbilical vein endothelial cells (HUVEC) are primary pooled cells that were obtained from Clonetics (Lonza, Switzerland). HUVEC were cultured in endothelial growth medium (EGM-2, Lonza, Switzerland), composed of endothelial basal medium (EBM-2, Lonza, Switzerland) and the SingleQuot Bullet Kit (Lonza, Switzerland). The cells were seeded at a density of 5000/cm2 in T75 flasks (Corning Costar, Sigma Aldrich, St. Louis, MO, USA).
3
Culturing Human Umbilical Vein Endothelial and THP-1 Monocytic Cells
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HUVECs, primary human umbilical vein endothelial cells, obtained from a pool of donors, were purchased from Clonetics (Lonza, Switzerland) and cultured in endothelial basal medium (EBM-2, CC-3156, Lonza) supplemented with SingleQuot Bullet Kit (CC-4176, Lonza) containing 0.1% human recombinant epidermal growth factor (rh-EGF), 0.04% hydrocortisone, 0.1% vascular endothelial growth factor (VEGF), 0.4% human recombinant fibroblast growth factor (rh-FGF-B), 0.1% insulin-like growth factor-1 with the substitution of arginine for glutamic acid at position 3 (R3- IGF-1), 0.1% ascorbic acid, 0.1% heparin, 0.1% gentamicin and amphotericin-B (GA-1000), and 2% fetal bovine serum (FBS). The cells were seeded at a density of 5000/cm2 in T75 flasks (Corning Costar, Sigma Aldrich, St. Louis MO, USA).
Human monocytic THP-1 cells were purchased from ATCC (Rockville, MD, USA) and maintained in RPMI-1640 medium supplemented with 2-mercaptoethanol to a final concentration of 0.05 mM and with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine (all from Euroclone, Milano, Italy). The cells were seeded at a density of 2 × 105 cells/ml in T75 flasks.
Human monocytic THP-1 cells were purchased from ATCC (Rockville, MD, USA) and maintained in RPMI-1640 medium supplemented with 2-mercaptoethanol to a final concentration of 0.05 mM and with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine (all from Euroclone, Milano, Italy). The cells were seeded at a density of 2 × 105 cells/ml in T75 flasks.
4
Culturing Human Vascular Cells
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Human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (NHDFs) were purchased from Clonetics (Lonza, Basel, Switzerland) and cultured in Endothelial Cell Growth Medium (EBM-2, CC-3156, Lonza) and Fibroblast Growth Basal Medium (FBM-2, CC-3131, Lonza) supplemented with endothelial and fibroblast SingleQuot Bullet Kit (CC4176 and CC-3132, Lonza), respectively. The HUVEC and NHDF cells were both seeded and sub-cultured before reaching confluence (70–80%) at a density of 5000/cm2 in a humidified atmosphere of 5% CO2 at 37°C. All cells tested negative for mycoplasma infection. Population doublings (PDs) were calculated by the formula: (log10F − log10I)/log102, where F is the number of cells at the end of the passage, and I is the number of seeded cells.
5
LPS-induced Inflammation in RAW264.7 and HUVEC Cells
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RAW264.7 cells (ATCC number: TIB-71) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) under humidified 5% CO2 at 37°C. Human umbilical vein endothelial cells (HUVEC) (KeyGen, KG419) were maintained in EBM-2 medium (Lonza) supplemented with SingleQuot Bullet Kit (Lonza) under humidified 5% CO2 at 37°C. For the LPS model, the cells were inoculated in a six-well plate and treated with different concentrations of PF 24 h later (10−4, 10−5, 10−6, and 10−7 M), 4 h before stimulation with LPS (Sigma, 1 μg/ml), and 12 h after stimulation with LPS, the protein of cells was extracted from the six-well plate.
6
HUVEC Response to Modified LDLs
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Cryopreserved HUVECs obtained from a pool of three donors were purchased from Clonetics (CC-2519, Lonza, Basel, Switzerland) and maintained in EBM-2 (CC-3156, Lonza) supplemented with SingleQuot Bullet Kit (CC-4176, Lonza) and maintained in a humidified atmosphere of 5% CO2 at 37 °C. Cells were seeded in flasks up to passage five at a density of 5000/cm2 and sub-cultured when they reached 70–80% confluence. All cell cultures were regularly tested for mycoplasma contamination.
8 × 105 HUVECs were cultured in 6-well plates and allowed to attach overnight before the treatment. Then, cells were exposed to buffer (lipoprotein free, negative control) or incubated with untreated LDLs, oxidized LDLs (ox-LDLs), or LDLs treated with sphingomyelinase (SMase-LDLs). Before incubation with cells, all LDL samples were passed in 0.2 µm filters under sterile conditions. Cells were treated in absence or in the presence of control or modified LDLs (20 µg/mL and 50 µg/mL) for 24 h; cells were then harvested and used for subsequent analysis [55 (link)].
8 × 105 HUVECs were cultured in 6-well plates and allowed to attach overnight before the treatment. Then, cells were exposed to buffer (lipoprotein free, negative control) or incubated with untreated LDLs, oxidized LDLs (ox-LDLs), or LDLs treated with sphingomyelinase (SMase-LDLs). Before incubation with cells, all LDL samples were passed in 0.2 µm filters under sterile conditions. Cells were treated in absence or in the presence of control or modified LDLs (20 µg/mL and 50 µg/mL) for 24 h; cells were then harvested and used for subsequent analysis [55 (link)].
7
Isolation and Culture of Airway Smooth Muscle and Epithelial Cells
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Pure primary ASM bundles were isolated from bronchial biopsies and from lung resection material. ASM cells were cultured in DMEM with glutamax-1 supplemented with 10% FBS, penicillin (100U/ml), streptomycin (100µg/ml), amphotericin (0.25µg/ml), 100µM nonessential amino acids, and 1mM sodium pyruvate (Gibco).
ASM was cultured and characterized for a-Smooth Muscle Actin (SMA) expression using a mouse monoclonal anti-SMA antibody (clone 1A4, Dako) or mouse IgG2a isotype control (clone DAK-G05, Dako) by flow cytometry and used between passage 2 to 6. Basal epithelial cells obtained from nasal brushings and bronchoscopy were grown on collagen (Advanced Bio Matrix, San Diego, California, USA) coated 12-well plates in bronchial epithelial growth medium (BEGM; Lonza, Berkshire, UK) including supplement SingleQuot BulletKit (Lonza), 0.3% Fungizone antimycotic (Invitrogen), and 1% antibiotic-antimycotic. The epithelial cells were expanded onto collagencoated T75cm 2 flasks and replenished with fresh medium three times a week.
ASM was cultured and characterized for a-Smooth Muscle Actin (SMA) expression using a mouse monoclonal anti-SMA antibody (clone 1A4, Dako) or mouse IgG2a isotype control (clone DAK-G05, Dako) by flow cytometry and used between passage 2 to 6. Basal epithelial cells obtained from nasal brushings and bronchoscopy were grown on collagen (Advanced Bio Matrix, San Diego, California, USA) coated 12-well plates in bronchial epithelial growth medium (BEGM; Lonza, Berkshire, UK) including supplement SingleQuot BulletKit (Lonza), 0.3% Fungizone antimycotic (Invitrogen), and 1% antibiotic-antimycotic. The epithelial cells were expanded onto collagencoated T75cm 2 flasks and replenished with fresh medium three times a week.
8
Culturing Normal Human Bronchial Epithelial Cells
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Normal HBECs were purchased from Lonza (Basel, Switzerland). Cells were cultured as a monolayer in bronchial epithelial cell basal medium supplemented with the Single Quot BulletKit (Lonza) at 378C in a humidified atmosphere in 5% CO 2 . Medium was changed every second day. For further details, see the Methods section in this article's Online Repository.
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