A murine prostate cancer cell line RM‐1 was purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China), and the HUVECs were obtained from iCell Bioscience (Shanghai, China). RM‐1 cells were cultured in basic Dulbecco's modified eagle medium (DMEM, Gibco, USA) containing high‐content glucose (4.5 g L−1) and 1 mm sodium pyruvate, supplemented by 10% v/v fetal bovine serum (FBS, Biological Industries, Israel) and 1% w/v penicillin/streptomycin (P/S). HUVECs were cultured in DMEM/Ham's F‐12 (1:1 mixed) with 2.5 mm L‐glutamine and 15 mm 2‐[4‐(2‐Hydroxyethyl)‐1‐piperazinyl]‐ethanesulfonic acid (DMEM/F‐12, Corning, USA), supplemented by 10% FBS and 1% P/S. Normally, RM‐1 cells and HUVECs were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air with an O2 concentration of about 21%. A hypoxic condition for incubating RM‐1 cells was created in a small tris‐gas incubator (Galaxy 48 R, Eppendorf, Germany) under 5% CO2, 94% N2, and only 1% O2. Both the hypoxic extracellular and intracellular microenvironments were achieved by preincubating cells in the hypoxic incubator for at least 12 h.
Huvecs
Manufactured by Corning
Sourced in United States
HUVECs (Human Umbilical Vein Endothelial Cells) are primary cells derived from the endothelial lining of human umbilical veins. They are used as an in vitro model for the study of endothelial cell biology and function.
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Lab products found in correlation
Huvecs, by Lonza (12 mentions)
Matrigel, by Corning (6 mentions)
Egm 2, by Lonza (5 mentions)
Dmem f12, by Corning (3 mentions)
Dulbecco modified eagle medium (dmem), by Corning (3 mentions)
Ebm 2, by Lonza (3 mentions)
Egm 2 media, by Lonza (3 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Tcl buffer, by Qiagen (2 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Accutase, by Innovative Cell Technologies (2 mentions)
Huvecs, by Procell (2 mentions)
Hct116, by American Type Culture Collection (2 mentions)
Dme 4t1 and 293t, by American Type Culture Collection (2 mentions)
Growth factor reduced matrigel, by Corning (2 mentions)
Huvecs, by Merck Group (2 mentions)
Egm 2 bulletkit, by Lonza (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Galaxy 48r, by Eppendorf (1 mentions)
35 protocols using huvecs
1
Murine Prostate Cancer Cell Culture
2
HUVEC Tube Formation Assay
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Human Umbilical Vein Endothelial Cells (HUVECs) (Corning) were used for tube formation, as these tubules were developed from clear elongated cell bodies that connect to form polygon networks. HUVECs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) basal medium (Lonza, Basel, Switzerland) supplemented with EGM-2 supplements (Lonza) and 1% penicillin/streptomycin. Harvested HUVECs were then mixed with conditioned media obtained from MDA-MB-231 after 24 h of exposure to BITC and/or SOR treatments and plated on basement membrane matrix (Corning® Matrigel® Matrix). Next, 2.5 × 104 HUVECs were used for each well and incubated with the conditioned media for 12–16 h at 37 °C. Images of the tube networks were taken by Zeiss microscope and further quantified using ImageJ 1.54 with the Angiogenesis Analyzer plugin [56 (link)].
3
3D Cellular Spheroid Fabrication
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HMSCs (Lonza, Walkersville, MD) and HUVECs (Lonza) were used to fabricate of 3D cellular spheroids. HMSCs were cultured in all-in-one ready-to-use hMSC growth medium (Cell Applications, INC., San Diego, CA). HUVECs or tdTomato + HUVECs were cultured in MCDB 131 medium (Corning, New York, NY) supplemented with 10% fetal bovine serum (Corning, New York, NY), 2 mM glutamine (Thermo Fisher Scientific, Waltham, MA), 1% penicillin/streptomycin (Corning, New York, NY), 4.5 ug/mL bovine brain extract (Lonza, Walkersville, MD), 10 unit/mL heparin (Sigma-Aldrich, St. Louis, MO), and 1.86 mg/mL endothelial cell growth supplement (Sigma-Aldrich, St. Louis, MO). Cells passages from three through seven were used for both HMSCs and HUVECs. The cells were expanded at 37 °C with 5% CO 2 in a humidified sterile incubator.
4
Infection Kinetics of HUVECs
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HUVECs were cultured as described previously [54 (link)]. Briefly, HUVECs (Cell Application, San Diego, CA) were cultivated in Prigrow I medium supplemented with 10% (vol/vol) heat-inactivated FBS in 5% (vol/vol) CO2 at 37°C. All experiments were performed between passages 5 and 7, and cells were maintained in Prigrow I medium with 3% (vol/vol) FBS. When HUVECs were confluent, they were collected and seeded onto 24-well plates (Corning Inc., Corning, NY). Once all wells were confluent, the HUVEC monolayers were infected with either 3 MOI, 10 MOI, or media only. Total RNA was extracted from each plate at 3, 24, and 48 hours post-infection (hpi) by using an RNeasy mini kit (Qiagen, Valencia, CA) and digested with RNase-free DNase (Qiagen). Gene expression was determined as described below. Cell-free culture supernatants were collected and stored in -80°C until protein analysis.
5
Culturing Human Cell Lines for Experimental Studies
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Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Walkersville, MD, USA) and grown at 37°C and 5% CO2 in EGM™ BulletKit™ medium (Lonza, CC‐3162: EBM2 + FBS, Hydrocortisone, hFGF, VEGF, R3‐IGF, Ascorbic acid, hEGF, Heparin, GA‐1000) according to the manufacturer's recommendation. The HUVECs were passaged by trypsinization (0.25%, Corning, 25‐053‐CI, NY, USA), and only passages 3–5 were used for experiments. Human melanoma MDA‐MB‐435 and human embryonic kidney 293T (HEK 293T) cell lines were grown in DMEM (Corning, 10‐013‐CVR); human TERT‐Retinal Pigment Epithelium 1 (RPE1) cells were cultured in DMEM F‐12 (Corning, 10‐090‐CVR); and human ductal breast epithelial tumor cell line T47D and mouse primary lung epithelial tumor cell line TC‐1 were maintained in RPMI 1640 (corning, 15‐040‐CVR) at 37°C and 5% CO2. All of cell culture media were supplemented with 10% fetal bovine serum (FBS) (Corning, 35‐016‐CV) and 1% penicillin–streptomycin (PS) (Corning, 30‐002‐CI).
6
HUVEC and HRE Co-Culture for RNA-Seq
7
HUVEC Permeability Assay with Histone H3 Peptides
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Human umbilical vein endothelial cells (HUVECs) (Lonza, Walkersville, MD, USA) were cultured using endothelial cell growth medium (EGM) BulletKit (Lonza, Walkersville, MD, USA). HUVECs (5 × 105 cells/ml) were grown on 12-mm Transwells with 0.4 μm pore polyester membrane inserts (Corning Life Sciences, Corning, NY, USA) for 3 days to develop a confluent (90%) monolayer. HUVECs were then treated for 16 h with 5 μg/ml histone H3 peptide (ARTKQTARKSTGGKAPRKQLATKAARKSAP) or CitH3 peptide [A(Cit)TKQTA(Cit) KSTGGKAP(Cit)KQLATKAA(Cit)KSAP]. Chambers were then incubated in the presence of 1 mg/ml 10-kDa FITC-dextran (Thermo Scientific, Rockford, IL, USA). The fluorescence of media in the lower chambers was measured by a GloMax-multi detection system (Promega, Madison, WI, USA).
8
Wound Healing and Migration Assays with HUVEC and BMSC-derived Exosomes
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For wound healing assays, human umbilical vein endothelial cells (HUVECs; ATCC) (4 × 104/well) were added to the lower chamber of a well containing a Transwell assay insert (8-μm pores, Corning, United States), with 20 μg of BMSCs-exo or C-BMSCs-exo being added into the upper compartment. The monolayer was scratched with a sterile pipette tip, washed with PBS, and cells were then cultured for 24 h in high-glucose DMEM, with cells being imaged at 0, 12, and 24 h via inverted microscope (Olympus Microscopes, Tokyo, Japan). Wound closure in six randomly selected fields of view was then quantified using Photoshop (Adobe Systems Inc., CA, United States), with data being normalized to mean values in the control group.
For migration assays, HUVECs (4 × 104) were instead added to the upper chamber of the Transwell insert, with BMSCs-exo or C-BMSCs-exo (20 μg) being added in the lower chamber. Following incubation for 18 h at 37°C, cells that had migrated to the lower chamber were fixed, stained with 0.5% crystal violet, and quantified via microscopy.
For migration assays, HUVECs (4 × 104) were instead added to the upper chamber of the Transwell insert, with BMSCs-exo or C-BMSCs-exo (20 μg) being added in the lower chamber. Following incubation for 18 h at 37°C, cells that had migrated to the lower chamber were fixed, stained with 0.5% crystal violet, and quantified via microscopy.
9
Angiogenesis Assay with Tumor Conditioned HUVEC
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Tube formation was observed in human umbilical vein endothelial cells (HUVECs) (354151; Corning Incorporated, New York, NY, USA) to evaluate angiogenesis. HUVECs and Y79 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and L-15 medium, respectively, in an incubator at 37°C. The Y79 cells were transfected, and the supernatant was collected 48 hours later. The cell debris was removed by centrifugation under aseptic conditions, and the tumor cell culture supernatant was obtained. Tumor conditioned medium was prepared using the mixture of tumor supernatant, DMEM, and FBS (4:5:1). Subsequently, each well in a 96-well plate was added with 50 µL Matrigel and allowed to polymerize in an incubator at 37°C for 30 minutes. HUVEC suspension was cultured with the prepared tumor conditioned medium at 37°C with 5% CO2 for 8 hours. Three replicates were set for each group. Afterward, four fields were randomly selected per well under phase contrast microscope, and the number of tubules was counted.
10
HUVEC Tube Formation Assay
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Human umbilical vein endothelial cell line (HUVEC) was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and incubated in Endothelial Cell Growth Medium BulletKit (EGM, Lonza, Switzerland) at 37°C with 5% CO2 in a humidified atmosphere. HUVECs (2 × 104) in 200 μL conditional medium from A549 and H460 cells were inoculated into a 24-well plate which was precoated with Matrigel (Corning) followed by incubation with 5% CO2 at 37°C for 6 h. Tube structures were photographed under a bright-field microscope (Nikon). The mesh and length of the completed tubes were measured to quantify tube formation using Image View 3.7 (Jingtong, China).
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