Huvec cells

Manufactured by American Type Culture Collection

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HUVEC (Human Umbilical Vein Endothelial Cells) are primary cells derived from the human umbilical vein. They are a commonly used in vitro model for the study of endothelial cell biology.

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HUVECs (892 citations) HUVEC (388 citations) Human umbilical vein endothelial cells (HUVECs) (34 citations) HUVEC cell line (26 citations) HUVECs cells (2 citations) Primary human umbilical vein endothelial cells (HUVECs) (2 citations)

38 protocols using huvec cells

Characterization of Glioblastoma Cell Lines and Conditioned Media

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GBM6 (Mayo Clinic), GBM43 (Mayo Clinic), DBTRG-05MG (ATCC), U-251 (ATCC), T98-G (ATCC), and LN-229 (ATCC) GBM cells; HUVEC cells (ATCC); THP-1 human monocytes (ATCC); and human astrocytes (ScienCell) were verified using short tandem repeat (STR) profiling, passaged under 6 times, and confirmed mycoplasma free. Breast CAFs were provided by the Breast Cancer Now Tissue Bank (London, United Kingdom). GBM cells were cultured in DMEM/F-12 plus 10% FBS and 1% penicillin/streptomycin at 37°C. HUVECs were grown in EGM-2 media (Lonza, catalog CC-3162). THP-1 cells were grown in complete RPMI with HEPES. human astrocytes were grown in Gibco Astrocyte Medium (Thermo Fisher).
To generate GSC-containing neurospheres, GBM cells were grown in NM, consisting of DMEM/F12 (Gibco, Thermo Fisher Scientific) supplemented with 20 ng/ml EGF (Peprotech), 20 ng/mL bFGF (Peprotech), and 2% GEM21/neuroplex (Gemini Bio-Products). When comparing CAF_CM to NM, CAF_CM was generated by replacing the media of cultured CAFs with NM for 72 hours, after which media was collected and centrifuged at 300g for 5 minutes, followed by filtration through a 40 μm filter.

Jain S., Rick J.W., Joshi R.S., Beniwal A., Spatz J., Gill S., Chang A.C., Choudhary N., Nguyen A.T., Sudhir S., Chalif E.J., Chen J.S., Chandra A., Haddad A.F., Wadhwa H., Shah S.S., Choi S., Hayes J.L., Wang L., Yagnik G., Costello J.F., Diaz A., Heiland D.H, & Aghi M.K. (2023). Single-cell RNA sequencing and spatial transcriptomics reveal cancer-associated fibroblasts in glioblastoma with protumoral effects. The Journal of Clinical Investigation, 133(5), e147087.

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Proliferation Assay of HUVEC Cells

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HUVEC cells were purchased from ATCC (Manassas, VA) and cultured as previously described30 (link). No further authentication was performed. Cells were seeded at 6.5 × 10⁴ cells per well in 100 μL of medium per well. Drugs were diluted in prewarmed DMEM. Serial dilutions were performed from 1000–0.1 nM. The cells were left to grow for 18 hours with exposure to vehicle control (DMSO) or drugs. Experiments were done in triplicate each time. Cellular proliferation was assessed using a CCK-8 assay (Dojindo, Rockville, MD). All plates were read at 450 nm.

Beedie S.L., Mahony C., Walker H.M., Chau C.H., Figg W.D, & Vargesson N. (2016). Shared mechanism of teratogenicity of anti-angiogenic drugs identified in the chicken embryo model. Scientific Reports, 6, 30038.

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TCDD Modulation of VEGFR1 Phosphorylation

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To check the phosphorylation of TCDD with hVEGFR1, we performed western blot analysis. HUVEC cells were purchased from ATCC (Manassas, VA) and cultured using endothelial cell growth medium with VEGF (ATCC# PCS-100–041). When cells reached near 80% confluence, they were incubated in DMEM medium without serum or growth factor for 24 hr. Then 10% serum was added to the medium as a positive control to stimulate VEGFR1 phosphorylation. Cells treated with vehicle (DMSO) was used as a negative control. TCDD with the indicated concentration was added to other samples. After 10 min treatment, cells were washed with PBS and lysed with RIPA buffer (Thermo Fisher). The phosphorylated VEGFR1 was determined by Western blot using anti-phospho-VEGFR1 (PTYR1333) antibody (Sigma). Tubulin was used as a loading control.

Chitrala K.N., Yang X., Busbee B., Singh N.P., Bonati L., Xing Y., Nagarkatti P, & Nagarkatti M. (2019). Computational prediction and in vitro validation of VEGFR1 as a novel protein target for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Scientific Reports, 9, 6810.

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Tumor Cell Transmigration Assay

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TEM assay was performed using the CytoSelect Tumor TEM assay kit (CBA-216, Cell Biolabs, Inc. San Diego, CA, USA). Briefly, HUVEC cells (25,000–50,000) (CRL-2922, ATCC, Manassas, VA, USA) were allowed up to 72 hours to form a monolayer on the upper surface of the membrane inside the insert and then activated by overnight serum starvation or TNFα treatment. The 10A-iK8 or 231-K8ikd cells (50,000) were incubated with the Cytotracker dye for 1 hour prior to further incubation in the insert on top of the HUVEC monolayer in the presence or absence of doxycycline. Depending upon specific experimental need, AMD3100 (35 ng/ml) was included to inhibit CXCR4 prior to and during the 22-hour assay time. The cells that migrated through the HUVEC monolayer were lysed and quantified using a Fluorescence multi-plate reader (PolarStar Omega, BMG LabTech, Cary, NC, USA).

Mukherjee D., Lu H., Yu L., He C., Lahiri S.K., Li T, & Zhao J. (2016). Krüppel-like factor 8 activates the transcription of C-X-C cytokine receptor type 4 to promote breast cancer cell invasion, transendothelial migration and metastasis. Oncotarget, 7(17), 23552-23568.

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Culturing Colorectal Cancer Cell Lines

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All CRC cell lines (SW480, SW620, HT-29, COLO 205, and WiDr) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in Dulbecco's Modified Eagle Medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 10 U/mL of penicillin-streptomycin (Hyclone). All cell lines were routinely tested for mycoplasma contamination and cell lines used in subsequent experiments (SW480 and SW620) were authenticated by short tandem repeat profiling at the Research Institute of National Cancer Center (Republic of Korea).
HUVEC cells were purchased from ATCC, and cultured in Endothelial Cell Growth medium (EGM-2; Lonza, Walkersville, MD, USA) supplemented with 10% FBS.

Park P.G., Jo S.J., Kim M.J., Kim H.J., Lee J.H., Park C.K., Kim H., Lee K.Y., Kim H., Park J.H., Dong S.M, & Lee J.M. (2017). Role of LOXL2 in the epithelial-mesenchymal transition and colorectal cancer metastasis. Oncotarget, 8(46), 80325-80335.

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Culturing Diverse Cancer Cell Lines

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CT26 cells (murine CRC cells), HCT116 cells (human colorectal cancer cells), DC2.4 cells (mouse dendritic cell line ), and HUVEC cells (human umbilical vein endothelial cells) were acquired from ATCC. These cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium at 37 °C in a humidified atmosphere with 5% CO₂. The RPMI‐1640 medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin.
For the purpose of in vivo studies, BALB/C mice (female, 6–8 weeks old) were purchased from the Shanghai Wushi Experimental Animal Center (Shanghai, China). All animal studies were approved by the Institutional Animal Care and Use Committee at Fujian Medical University.

Wang L., Zhou H., Chen Q., Lin Z., Jiang C., Chen X., Chen M., Liu L., Shao L., Liu X., Pan J., Wu J., Song J., Wu J, & Zhang D. (2023). STING Agonist‐Loaded Nanoparticles Promotes Positive Regulation of Type I Interferon‐Dependent Radioimmunotherapy in Rectal Cancer. Advanced Science, 11(7), 2307858.

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Female Mice Study: HUVEC Cultivation

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Female C57BL/6 mice (8 to 12 weeks old) were purchased from Changzhou Cavens Laboratory Animal Ltd. (Changzhou, China). All experiments were approved by the Ethical Committee for Animal Experiments of Fudan University, and strictly carried out in accordance with the approved guidelines. HUVEC cells were purchased from ATCC (Manassan, VA), and maintained in RPMI 1640 containing 10% foetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA).

Fang W., Wang G., Tang L., Su H., Chen H., Liao W, & Xu J. (2018). Hydrogen gas inhalation protects against cutaneous ischaemia/reperfusion injury in a mouse model of pressure ulcer. Journal of Cellular and Molecular Medicine, 22(9), 4243-4252.

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HUVEC Cell Culture Protocol

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HUVEC cells were purchased from ATCC cell bank. The cells were cultured in F‐12K medium and maintained at 37°C in the presence of 5% CO₂ incubator.

He Y., Wu Z., Qiu C., Wang X., Xiang Y., Lu T., He Y., Shang T., Zhu Q., Wang X., Zeng Q., Zhang H, & Li D. (2019). Long non‐coding RNA GAPLINC promotes angiogenesis by regulating miR‐211 under hypoxia in human umbilical vein endothelial cells. Journal of Cellular and Molecular Medicine, 23(12), 8090-8100.

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Survivin siRNA Transfection in Glioma Cells

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All oligonucleotides were synthesized and purified by Sangon Biotech Company (Shanghai, China). Survivin siRNA (5ʹ-AUUCACCAAGGGUUAAUUCdTdT-3ʹ) was synthesized by GenePharma Company (Shanghai, China). Deionized water used in all aqueous solutions was produced using a Millipore Ultrapure water machine (Massachusetts, USA). GelRed DNA gel stain solution was purchased from Biosharp (Beijing, China). Tris, magnesium chloride (MgCl₂), and Triton X-100 were obtained from Macklin (Shanghai, China). Agarose was purchased from Biowest (Barcelona, Spain). U87, U251, and HUVEC cells were purchased from ATCC. Fetal bovine serum (FBS) and high-glucose Dulbecco’s modified Eagle’s medium (DMEM/high glucose) were bought from Gibco (NY, USA). A cell counting kit (CCK-8) was purchased from Bimake (Texas, USA). 4′6-Diamidino-2-phenylindole (DAPI) were obtained from Solarbio (Beijing, China). Primary antibodies β-actin (20536-1-AP), NCL (10556-1-AP), and Caspase-3 (19677-1-AP) were obtained from Proteintech (IL, USA). Survivin (YT4472) was purchased from Immunoway (TX, USA), and horseradish peroxidase-conjugated or CoraLite594-conjugated secondary antibodies were purchased from Proteintech (IL, USA).

Zhou Y., Yang Q., Wang F., Zhou Z., Xu J., Cheng S, & Cheng Y. (2021). Self-Assembled DNA Nanostructure as a Carrier for Targeted siRNA Delivery in Glioma Cells. International Journal of Nanomedicine, 16, 1805-1817.

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Phycocyanin Extraction and Cell Analysis

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The crude extract of R-phycocyanin was prepared on a laboratory scale from Porphyra haitanensis (the contents of crude protein 15.78 μg/mL). HUVEC cells were purchased from ATCC. Drosophila melanogaster was from Engineering Research Centre of Fujian-Taiwan Special Marine Food Processing and Nutrition.
Senescence-associated β-galactosidase (SA β-gal) staining, Hoechst and PI Staining Kit, Annexin V Apoptosis Detection Kit, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Cells counting kit-8 (CCK-8), the Mitochondrial Membrane Potential Assay Kit with JC-1, DNA content quantitation assay, and Nuclear Protein Extraction Kit were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). ECL was purchased from Thermo Fisher Scientific (Waltham, MA, USA). All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Feng Y., Lu H., Hu J., Zheng B, & Zhang Y. (2022). Anti-Aging Effects of R-Phycocyanin from Porphyra haitanensis on HUVEC Cells and Drosophila melanogaster. Marine Drugs, 20(8), 468.

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