Primary human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HUVECs were cultured and grown in vascular cell basal medium (PCS‐100‐030; ATCC) supplemented with bovine brain extract (0.2%), recombinant human (rh) EGF (5 ng/ml), L‐glutamine (10 mM), heparin sulphate (0.75 IU/ml), hydrocortisone hemisuccinate (1 μg/ml), 2% FBS and ascorbic acid (50 μg/ml). All supplements were purchased from ATCC (#PCS‐100‐040). These cells were cultured at 37°C in a CO2 humidified atmosphere and detached from the growth plates using non‐enzymatic cell dissociation solution (Cellstriper; Corning Costar).
Huvecs
Manufactured by American Type Culture Collection
Sourced in United States, China, Switzerland, United Kingdom, Canada
HUVECs (Human Umbilical Vein Endothelial Cells) are primary cells derived from the human umbilical vein. They are used as an in vitro model for the study of endothelial cell biology and function.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (177 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (103 mentions)
Huvecs, by Thermo Fisher Scientific (74 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (54 mentions)
Streptomycin, by Thermo Fisher Scientific (43 mentions)
Penicillin, by Thermo Fisher Scientific (41 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (38 mentions)
Huvecs, by Lonza (26 mentions)
Egm 2, by Lonza (24 mentions)
Huvecs, by Merck Group (24 mentions)
Endothelial cell medium (ecm), by ScienCell (23 mentions)
Rpmi 1640, by Thermo Fisher Scientific (23 mentions)
Heparin, by Merck Group (21 mentions)
Matrigel, by BD (18 mentions)
Mda mb 231, by American Type Culture Collection (17 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (17 mentions)
Streptomycin, by Merck Group (16 mentions)
Penicillin, by Merck Group (16 mentions)
Endothelial cell growth supplement (ecgs), by ScienCell (15 mentions)
Hct116, by American Type Culture Collection (14 mentions)
892 protocols using huvecs
1
Culturing Primary Human Umbilical Vein Endothelial Cells
2
ccRCC Primary Tumor Culture and ADRB Antagonist Assays
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Clear cell renal cell carcinoma primary tumor cultures (ccRCC) were obtained from surplus of resected surgery tumor samples from VHL patients following the procedure previously described (16), and cultured in RPMI medium supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin (all from GIBCO, Grand Island, NY, USA). All patients provided written informed consent to use their tissue samples for this study.
HUVECs (ATCC-CRL-1730) were cultured in EGM-2 (Lonza, Walkersville, MD, USA) supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin (GIBCO, Grand Island, NY, USA).
The human renal cancer cell line 786-O (ATCC CRL-1932) was cultured in RPMI supplemented with 20% FBS, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin.
ccRCC primary tumors, 786-O cells, and HUVECs were incubated with different doses of ADRB antagonists for the time and dose indicated in each experiment. Atenolol (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO (Merck, Darmstadt, Germany), while propranolol and ICI (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in distilled water.
All the cellular assays were performed at 37 °C, 5% CO2 and humidity conditions.
HUVECs (ATCC-CRL-1730) were cultured in EGM-2 (Lonza, Walkersville, MD, USA) supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin (GIBCO, Grand Island, NY, USA).
The human renal cancer cell line 786-O (ATCC CRL-1932) was cultured in RPMI supplemented with 20% FBS, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin.
ccRCC primary tumors, 786-O cells, and HUVECs were incubated with different doses of ADRB antagonists for the time and dose indicated in each experiment. Atenolol (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO (Merck, Darmstadt, Germany), while propranolol and ICI (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in distilled water.
All the cellular assays were performed at 37 °C, 5% CO2 and humidity conditions.
3
Culturing Human Endothelial Cell Lines
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Human umbilical vein endothelial cells (HUVECs) and EA.hy926 human endothelial-derived cell lines were purchased from American Type Culture Collection (ATCC, Manassas, FL, United States). HUVECs were cultured in vascular cell basal medium (ATCC) supplemented with endothelial cell growth kit–VEGF, including 5 ng/mL rh VEGF, 5 ng/mL rh EGF, 5 ng/mL rh FGF basic, 15 ng/mL rh IGF, 10 mM L-glutamine, 0.75 U/mL heparin sulfate, hydrocortisone 1 μg/mL, ascorbic acid 50 μg/mL, fetal bovine serum 2%, 10 U/mL penicillin, and 10 μg/mL streptomycin. EA.hy926 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (MilliporeSigma, St. Louis, MO, United States). Both cell lines were incubated at 37°C in 75 cm2 tissue culture-treated flasks in a 5% CO2 humidified incubator. Every other day, the media were replaced and the cells were subcultured at 80% confluence.
4
Cell Culture of Various Cell Lines
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C2C12 (CRL‐1772), HUVECs (CRL‐1730), HEK‐293 (CRL‐1573), and hMSCs (PCS‐500‐012) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). HDFs (PH10605A) were obtained from Genlantis (San Diego, CA). hMSCs were cultured in alpha MEM containing 10% fetal bovine serum (FBS, 35‐015‐CV, Corning, NY), 1% penicillin–streptomycin (P/S, 15140–122, Gibco, Grand Island, NY), and 2 mM GlutaMAX (35050‐061, Gibco). HUVECs were maintained in vascular cell basal medium (PCS‐100‐030, ATCC) supplemented with Endothelial Cell Growth Kit‐VEGF (PCS‐100‐041, ACTT). Other cell types, such as C2C12 (DMEM, Welgene, Inc., Deagu, Korea), HEK (DMEM, Welgene), and HDF (DMEM, Welgene), were cultured in appropriate cell culture media supplemented with 10% FBS and 1% P/S.
5
Culturing Human Umbilical Cord Vein Endothelial Cells
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Human umbilical cord vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC). HUVECs were cultured in vascular cell basal medium (PCS100030; ATCC) supplemented with vesicle-free endothelial cell growth kit components (PCS100041; ATCC) with the addition of 1% penicillin-streptomycin (PCS999002; ATCC) and 2% exosome-free fetal bovine serum (FBS) (EXO-FBSHI-250A-1; Systems Biosciences). HUVECs at passage 3 were cultured in 6-well plates until they reached 80% confluence.
6
In Vitro and In Vivo Cancer Models
7
Angiogenic Potential of RA-FLS Secretome
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HUVECs (1 × 104 cells/well; ATCC, USA) were seeded in 96-well plates coated with CeturegelTM Matrix LDEV-free Matrigel and incubated at 37°C for 1 h. The supernatant of RA-FLS after different treatments was added to the culture wells of HUVECs as conditioned medium. The 96-well plates were incubated for 6 h and photographed under an inverted microscope for tube formation visualization. Tube-forming capability was quantified by counting the total number of closed polygons formed.
8
Culturing Human Gastric Cancer and Endothelial Cells
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The human gastric cancer cell line AGS and human umbilical vein endothelial cells (HUVECs) were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). AGS cells were cultured in RPMI 1640 medium (GIBCO-BRL, Gaithesberg, MD) supplemented with 10% fetal bovine serum (FBS, GIBCO-BRL), 100 U/mL penicillin and 100 μg/mL streptomycin. HUVECs (ATCC) were cultured in F12K medium (ATCC) supplemented with 10% FBS (GIBCO-BRL), 0.1 mg/mL heparin sulfate, 0.05 mg/mL endothelial cell growth factor supplement (BD Bioscience, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were cultured in a 5% at 37°C in a humidified atmosphere of 5% CO2.
9
Uptake and Colocalization of Extracellular Vesicles in HUVECs
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EVs uptake was monitored using PKH26 fluorescent labeling kit (Sigma-Aldrich, CA, United States). EV sample was diluted in diluent C, and then PKH26 dye was added and kept at room temperature. After 4 min incubation avoiding light exposure, the reaction was neutralized by 5% bovine serum albumin. Then, EVs, resuspended in PBS were centrifuged at 100,000 g for 70 min, and incubated with HUVECs for 4 h. Subsequently, HUVECs (ATCC, VA, United States) were fixed with 4% paraformaldehyde, labeled with 4′,6-diamidino-2-phenylindole (0.5 μg/ml; Invitrogen) and imaged under a confocal microscope (Carl Zeiss, Oberkochen, Germany) (Zhang et al., 2021 (link)).
When HUVECs reached 50–60% confluence, they were incubated with dextran 498 (Dx498, 100 μg/ml) for 2 h to label cell membrane lysosomes, washed with PBS and cultured with complete culture medium for 18 h. Finally, HUVECs were incubated with PKH26-labeled EVs for colocalization analysis.
When HUVECs reached 50–60% confluence, they were incubated with dextran 498 (Dx498, 100 μg/ml) for 2 h to label cell membrane lysosomes, washed with PBS and cultured with complete culture medium for 18 h. Finally, HUVECs were incubated with PKH26-labeled EVs for colocalization analysis.
10
Apoptosis Analysis of HUVECs
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Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum in an incubator with 37°C and 5% CO2.
Apoptosis analysis of HUVECs was performed by flow cytometry according to manufacturer’s instructions. Briefly, treated or untreated HUVECs were collected and labeled with Annexin V-APC/7-AAD apoptosis detection kit (KeyGen Biotech, Jiangsu, People’s Republic of China), followed by flow cytometry analysis.
For p53 immunofluorescence, HUVECs were seeded on coverslips in a 24-well plate. After treating HUVECs with 0 (control), 30, and 60 μg/mL PEG-b-PCL nano-micelles for 24 hours, the cells were fixed with 4% PFA for 30 minutes, permeabilized with 0.1% Triton X–100 for 10 minutes, and blocked with 5% bovine serum albumin for 1 hour. After blocking, the cells were stained with p53 antibody (rabbit, Santa Cruz Biotechnology Inc, Dallas, TX, USA) overnight at 4°C, followed by 1 hour incubation with anti-rabbit secondary antibody and 4′,6-diamidino-2-phenylindole at room temperature. Samples were imaged using a fluorescence microscopy (Olympus BX81; Olympus).
Apoptosis analysis of HUVECs was performed by flow cytometry according to manufacturer’s instructions. Briefly, treated or untreated HUVECs were collected and labeled with Annexin V-APC/7-AAD apoptosis detection kit (KeyGen Biotech, Jiangsu, People’s Republic of China), followed by flow cytometry analysis.
For p53 immunofluorescence, HUVECs were seeded on coverslips in a 24-well plate. After treating HUVECs with 0 (control), 30, and 60 μg/mL PEG-b-PCL nano-micelles for 24 hours, the cells were fixed with 4% PFA for 30 minutes, permeabilized with 0.1% Triton X–100 for 10 minutes, and blocked with 5% bovine serum albumin for 1 hour. After blocking, the cells were stained with p53 antibody (rabbit, Santa Cruz Biotechnology Inc, Dallas, TX, USA) overnight at 4°C, followed by 1 hour incubation with anti-rabbit secondary antibody and 4′,6-diamidino-2-phenylindole at room temperature. Samples were imaged using a fluorescence microscopy (Olympus BX81; Olympus).
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