Pooled HUVEC (from 3 to 6 individual donors, Lonza, Cologne, Germany) and HUAEC (from 250 individual donors, PeloBiotech GmbH, Planegg, Germany) were cultivated in EGM-2 Bullet Kit containing endothelial cell culture medium supplemented with 2% (v/v) fetal bovine serum, vascular endothelial growth factor, basic fibroblast growth factor, human epithelial growth factor, insulin-like growth factor-1, hydrocortisone, heparin, ascorbic acid, gentamycin, and amphotericin B (Lonza) in a standard humidified incubator at 37 °C with 5% (v/v) CO2. Cells of passage 5 were seeded in 4-well PCA chamber slides (Sarstedt, Nürnbrecht, Germany) at a cell density of 15,000 cells/well with 1 mL medium per well, and cultivated for up to 7 days. Medium was exchanged at day 2 and day 4 of cultivation.
Pooled huvec
Manufactured by Lonza
Sourced in Germany, United States
Pooled HUVECs are a commercially available cell line derived from human umbilical vein endothelial cells. They are intended for use in in vitro cell culture experiments.
Automatically generated - may contain errors
Lab products found in correlation
Egm 2 bulletkit, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Heparin, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Basic fibroblast growth factor (bfgf), by Lonza (1 mentions)
Vascular endothelial growth factor (vegf), by Lonza (1 mentions)
Human epithelial growth factor, by Lonza (1 mentions)
Insulin like growth factor 1, by Lonza (1 mentions)
Gentamycin, by Lonza (1 mentions)
Amphotericin b, by Lonza (1 mentions)
Hydrocortisone, by Lonza (1 mentions)
Ascorbic acid, by Lonza (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Human umbilical vein endothelial cells (huvec), by BD (1 mentions)
Egm singlequot, by Lonza (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Cellr microscope, by Olympus (1 mentions)
13 protocols using pooled huvec
1
Endothelial Cell Culture and Characterization
2
Transfection of Passage 3 HUVEC
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Pooled HUVEC were obtained from Lonza. Upon reception, HUVEC at P0 were expanded over 2 passages in collagen 1 (from rat tail, BD) coated flasks in complete EGM-2 medium supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a 5% CO2 -3% O2 humidified chamber. HUVEC at passage 3 were transfected twice at 24 h-intervals with 30 nM siRNA and Lipofectamine RNAi max (Life Technologies, ref.
3
Hypoxia Exposure in HUVEC and HeLa Cells
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Pooled HUVECs (Lonza) were cultured in endothelial basal medium containing EGM SingleQuots (Lonza) and 10% fetal calf serum (Invitrogen); HeLa cells in DMEM (Gibco) with 10% fetal calf serum and 1% penicillin/streptomycin. All cells tested negative for mycoplasma. Hypoxia was induced by incubation at 0.2% O2 for 12 h or 24 h.
4
Anregulatory Epidermal Growth Factor-Induced Angiogenesis
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Pooled HUVECs were purchased from Lonza and cultured according to the supplier's instructions. For the Matrigel tube-formation assay, reduced growth factor Matrigel™ (BD Biosciences, Bedford, MA, USA) was thawed overnight at 4°C. The Matrigel was allowed to solidify in 48-well culture dishes at 37°C for 30 min. The cells were harvested and seeded at a density of 2×105 cells/well in the presence or absence of recombinant AREG. In separate experiments, the cells were either treated with an AREG-neutralizing antibody (R&D Systems, Minneapolis, MN) or conditioned medium collected from MCF7-shCTL and MCF7-shGPR81 cells. The cells were then incubated at 37°C for an additional 12 h. Tube formation was observed by capturing images using an Olympus CellR microscope. The Matrigel assay results were quantified by measuring the total number of pixels in thresholded images using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). Three independent experiments were performed, and each experiment was performed in triplicate. Student's t-test was performed to determine the significance between the test groups.
5
Primary Human Umbilical Vein Endothelial Cell Culture
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Pooled HUVECs were purchased from Lonza (Walkersville, MD). Cells were expanded for two passages and then frozen and stored in liquid nitrogen. Cells were cultured in EGM2 growth medium (Lonza) containing 2% (vol/vol) fetal bovine serum (FBS). Cells were used between passages 5 and 10. All experiments were performed by seeding at 8 × 104 cells/cm2, allowing for cells to be confluent immediately after seeding.
6
Pravastatin's Effects on HUVECs
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Human umbilical vein ECs (Pooled HUVECs, Lonza) were grown in the EC growth medium supplemented with growth factors, 5% fetal bovine serum (FBS), and antibiotics (EGMTM‐2 BulletkitTM; Lonza) at 37oC and 5% CO2. Passage 4–6 HUVECs were used for the experiments. Cells were (60%–70%) confluent and were starved over‐night in MCDB‐131 basal medium with 1% FBS, followed by treatment with pravastatin sodium salt hydrate (10 µM; Sigma) (Abe et al., 2006 ; Panczel et al., 2019 ) in the same MCDB‐131 basal medium with 1% FBS for 24 hr. Controls were treated with vehicle phosphate‐buffered saline. To validate the expression level of top up‐ or downregulated lncRNAs and mRNAs, HUVECs were cultured and treated as previously described.
7
Synovial Fibroblast Culture and Characterization
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Synovial fibroblasts were cultured from the synovial tissue samples of four patients (24, 24, 28 and 30 years-old) from the OA groups, as previously described (21 (link)). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS) (both from Thermo Fisher Scientific Inc., Beijing, China), in a humidified atmosphere containing 95% air and 5% CO2 at 37°C. Cell passages 3–8 were used in the present study. Pooled HUVECs were purchased from Lonza (Walkersville, MD, USA), and grown in endothelial growth medium 2 (Lonza), with passages 3–8 being used in the present study. IL-1β, DKK-1, human DKK-1 affinity purified rabbit polyclonal antibody (DKK-1 inhibitor), and recombinant human DKK-1 (rhDKK-1) were purchased from R&D systems, Inc. (Minneapolis, MN, USA). The rabbit monoclonal anti-HIF-1α (Epitomics Inc., Burlingame, CA, USA; ab190197; 1:1,000) and rabbit polyclonal anti-VEGF antibodies (GeneTex Inc., Irvine, CA, USA; GTX102643; 1:1,000) were obtained from Cell Signaling Technologies, Inc. (Danvers, MA, USA). Rabbit monoclonal DKK-1 was purchased from Abcam (Abcam, Cambridge, CA, USA; ab109416; 1:1000). Anti-GAPDH was purchased from Santa Cruz Biotechnology Biotechnology, Inc. (Dallas, TX, USA).
8
Quantifying PUUV Virus Infectivity in HUVECs
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Briefly, Vero E6 cells (ATCC) were maintained in E-MEM with EBSS (BioWhittaker), and 10% FBS and infected at 0.01 MOI with the PUUV strain Sotkamo. After 14 days of infection, cell-free virus was harvested and stored at -80°C.
Pooled HUVECs (Lonza) were cultured using the EGM-2 BulletKit (Lonza) according to the manufacturer’s instructions. Prior to infection, the cells were cultured without hydrocortisone. For viral titration, 1.5x104 cells were seeded in 96-well plates and incubated at 37°C with 50μL of 1:10 serially diluted viral concentrations for three days. Subsequently, cells were fixed with 80% acetone for 10 min and stained with mouse anti-hantavirus mAb A1C5 (1:100, Progen) and AF488 conjugated goat anti-mouse mAb (1:400, Invitrogen). Cell nuclei were counterstained with DAPI and counted using LAS X software (Leica Microsystems). Viral titers were calculated as infected cells per μL virus containing supernatant.
Pooled HUVECs (Lonza) were cultured using the EGM-2 BulletKit (Lonza) according to the manufacturer’s instructions. Prior to infection, the cells were cultured without hydrocortisone. For viral titration, 1.5x104 cells were seeded in 96-well plates and incubated at 37°C with 50μL of 1:10 serially diluted viral concentrations for three days. Subsequently, cells were fixed with 80% acetone for 10 min and stained with mouse anti-hantavirus mAb A1C5 (1:100, Progen) and AF488 conjugated goat anti-mouse mAb (1:400, Invitrogen). Cell nuclei were counterstained with DAPI and counted using LAS X software (Leica Microsystems). Viral titers were calculated as infected cells per μL virus containing supernatant.
9
Culturing Human Endothelial Cells and Malaria Parasites
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Pooled HUVECs were obtained from Lonza (Walkersville, MD). HUVECs were grown in endothelial basal medium 2 (EBM-2) supplemented with EGM-2 SingleQuots (Lonza). Cells were maintained at 37°C and 5 percent CO2. HUVECs were used before passage six. P. falciparum 3D7 cells expressing green fluorescent protein (3D7HT-GFP) [26 (link)] were obtained from the Malaria Research and Reference Reagent Resource Center (MR4, http://www.mr4.org/). Cells were grown in P. falciparum culture medium, containing RPMI 1640 with 25 mM HEPES (Sigma-Aldrich, Saint Louis, MO) supplemented with 0.5 percent Albumax (GIBCO, Carlsbad, CA), sodium bicarbonate (2 g/l), 0.1 mM hypoxanthine, and gentamycin (50 μg/l). Albumax is an inexpensive substitute for human serum in P. falciparum culture medium, which has less batch-to-batch variation than does serum [27 (link)], although it may not be suitable for drug sensitivity studies [27 (link),28 (link)]. Albumax has been widely used for culture of P. falciparum. Human erythrocytes were obtained from Innovative Research (Novi, MI). Purified human RBCs were added to P. falciparum cultures at 2 percent hematocrit. Culture flasks were gassed with 90 percent N2, 5 percent O2, and 5 percent CO2 and incubated at 37°C.
10
Culturing Cos-7 and HUVEC Cells
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Cos-7 cells (ATCC, CRL-1651) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Corning, 10013CV) containing L-glutamine and sodium pyruvate and supplemented with 10% fetal bovine serum (Gibco, 10438-026) and 100 IU/ml penicillin-streptomycin (Gibco, 15070-063). Human Umbilical Vein Endothelial Cells (Pooled HUVECs, Lonza, C2519A) were cultured in Endothelial Cell Growth Medium-2 (EGM2, Lonza, CC-3156) supplemented with the BulletKit (Lonza, CC-3162). Cells were maintained at 37 °C and 5% CO2. Cells were passaged at 90% confluency, resuspended in fresh media, and one-fifth was plated on 100 mm Tissue Cell Culture Dishes (Falcon, 353003). For HUVECs full EGM-2 media was first placed onto a cell culture dish and left to equilibrate into the incubator for 30 min.
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