Huvecs

Manufactured by Keygen Biotech

Sourced in China

HUVECs are primary human umbilical vein endothelial cells. They are a commonly used in vitro cell model for studying endothelial cell biology and angiogenesis.

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HUVEC (3 citations) HUVEC-C (3 citations) HUVEC cell line (2 citations)

23 protocols using huvecs

Antioxidant Pretreatment Modulates PCB Exposure

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HUVECs and human monocytic cells (THP-1 cells) were purchased from KeyGEN Biotech Co., Ltd. HUVECs and THP-1 cells were grown in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd.),

100 units per milliliter

penicillin, and

100 micrograms per milliliter

streptomycin at 37°C in a 5%

{CO}_{2}

incubator. In addition, the medium for THP-1 cells was supplemented with

50 micromolar

β -mercaptoethanol

. HUVECs (

1 times 10 begin superscript 6 end superscript

) were seeded in six-well culture plates overnight and exposed to

5 micromolar

PCB29-pQ for 24 h for subsequent experimental testing. HUVECs were pretreated with antioxidants (

40 micromolar

VC,

20 micromolar

VE,

5 millimolar

NAC,

200 units per milliliter

PEG-SOD,

500 units per milliliter

PEG-CAT, or

5 millimolar

GSH-MEE) for 1 h, followed with

5 micromolar

PCB29-pQ exposure for 6 h for subsequent experimental testing. Parallel control cultures received equal volumes of dimethyl sulfoxide (DMSO). Each experiment was independently repeated at least three times.

Yang B., Ye Z., Wang Y., Guo H., Lehmler H.J., Huang R., Song E, & Song Y. (2022). Evaluation of Early Biomarkers of Atherosclerosis Associated with Polychlorinated Biphenyl Exposure: An in Vitro and in Vivo Study. Environmental Health Perspectives, 130(3), 037011.

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Human Trophoblast Cell Culture Protocols

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Four human trophoblast cell lines (HTR/SVneo, JAR, JEG3, and BeVo) and HUVECs were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). HTR/SVneo and JAR cells were cultured in RPMI1640, JEG3 cells in MEM, BeVo cells in F12K, and HUVECs in endothelial cell medium (ECM) (all media from KeyGEN, Nanjing, China) supplemented with 10% fetal bovine serum (FBS) (Gibco-BRL, Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen). All cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C.

Wu D., Xu Y., Zou Y., Zuo Q., Huang S., Wang S., Lu X., He X., Wang J., Wang T, & Sun L. (2018). Long Noncoding RNA 00473 Is Involved in Preeclampsia by LSD1 Binding-Regulated TFPI2 Transcription in Trophoblast Cells. Molecular Therapy. Nucleic Acids, 12, 381-392.

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HUVEC Culture and Conditioned Media Preparation

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Human umbilical vein endothelial cells (HUVECs) were obtained from Keygen biotech (Nanjing, China). Cells were maintained in DMEM:F12K medium (Gibco, GrandIsland, NY) containing 10% (v/v) fetal bovine serum (FBS) (Hyclone, Logan, UT), 100 units penicillin/mL, 100 mg/mL streptomycin,0.1mg/mL heparin and 0.05mg/mL endothelial cell growth supplement (ECGS) in a humidified atmosphere at 37°C with 5% CO₂. Confluent cultures between passages 2–3 were used in this study to minimize age-dependent variation in the level of apoptosis. Conditioned medium (CM) was collected in sterile conditions followed by centrifugation at 3000 rpm for 20 min at 4°C, and then stored at −80°C for further use. CM1 and CM2 are self conditioned media collected from the culture medium of HUVECs-transfected with empty vector and Mxi1-0 plasmid, respectively. Complete medium alone without cells was incubated under the same experimental conditions served as control.

Wu W., Hu Z., Wang F., Gu H., Jiang X., Xu J., Zhan X., Zheng D, & Zhang Z. (2017). Mxi1-0 regulates the growth of human umbilical vein endothelial cells through extracellular signal-regulated kinase 1/2 (ERK1/2) and interleukin-8 (IL-8)-dependent pathways. PLoS ONE, 12(6), e0178831.

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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).

Zhou T., Dong Q., Shen Y., Wu W., Wu H., Luo X., Liao X, & Wang G. (2016). PEG-b-PCL polymeric nano-micelle inhibits vascular angiogenesis by activating p53-dependent apoptosis in zebrafish. International Journal of Nanomedicine, 11, 6517-6531.

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Engineered Hydrogel for Vascular Cell Studies

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GelMA with photoinitiator, lithium phenyl‐2,4,6‐trimethylbenzoylphosphinate, was purchased from Intelligent Manufacturing Research Institute (IMRI, GM‐90, Suzhou, China). TNF‐α, ox‐LDL, and IL‐1β were purchased from Sigma‐Aldrich (St. Louis, USA). DAPI, HUVECs, human SMCs, and actin‐tracker green were purchased from Keygen Biotech Co., Ltd. (Nanjing, China). Paraformaldehyde (4% w/v), Triton X‐100, CCK‐8, and phosphate‐buffered saline (PBS) were purchased from Solarbio (Beijing, China). Mouse MCs were purchased from Kemao Biotechnology (Dongguan, China). Penicillin–streptomycin, Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), and endothelial basal medium (F‐12K) were purchased from Gibco (Grand Island, USA). Cell labeling kits (Qtracker 525 and 655), α‐SMA, VE‐Cad primary polyclonal antibody, and goat anti‐rabbit IgG (H+L) secondary antibody were obtained from Thermo Fisher (Waltham, USA).

Wang Y., Huang N, & Yang Z. (2023). Revealing the Role of Zinc Ions in Atherosclerosis Therapy via an Engineered Three‐Dimensional Pathological Model. Advanced Science, 10(18), 2300475.

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Endothelial Cell Injury Model Manipulation

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HUVECs were purchased from KeyGen BioTECH Corp., Ltd (Nanjing, China) and grown in DMEM (11995065, Thermo Fisher Scientific, San Jose, USA) with 5% CO₂ at 37℃. The model of endothelial cell injury was constructed with 10 ng/mL TNF-α for 24 h. Overexpression plasmids, pcDNA3.1-ALKBH5 and pcDNA3.1-Bcl2 were purchased from YouBio (Changsha, China). The siRNA-Bcl2 and siRNA-control were synthetized from Sangon Biotech (Shanghai, China). Lipofectamine 2000 (11668500, Thermo Fisher Scientific, San Jose, USA) was used for the transfection. miR-7 mimic (5′-TGGAAGACTAGTGATTTTGTTG-3′) and negative control (NC) mimic (5′-GGUUCGUACGUACACUGUUCA-3′) were synthetized from GeneChem Co., Ltd (Shanghai, China).

Zhang X., Deng S., Peng Y., Wei H, & Tian Z. (2022). ALKBH5 inhibits TNF-α-induced apoptosis of HUVECs through Bcl-2 pathway. Open Medicine, 17(1), 1092-1099.

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HUVEC Manipulation and TNF-α Response

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Human Umbilical Vein Endothelial Cells (HUVECs), obtained from Jiangsu KeyGEN BioTECH (KeyGEN, KG419), were cultured in Ham's F‐12K medium (Gibco, 21127030) with 1% Endothelial Cell Growth Supplement (ECGS) (KeyGEN, KGY1052), 1% penicillin‐streptomycin, 10% fetal bovine serum (Gibco, 10099141), and .1 mg/mL heparin (KeyGEN, KGY3149H) at 37°C in a humidified atmosphere containing 5% CO₂. After being seeded on appropriate culture plates overnight, HUVECs were exposed to 100 ng/mL tumour necrosis factor‐α (TNFα) (Novoprotein, C008) for different time periods.
Rictor gene knockout of HUVECs and corresponding control cells were generated using the CRISPR/Cas9 system obtained from Hanbio biotechnology. Rictor gene knockout of HUVECs were confirmed by sequencing and validated by western blotting. For Rictor overexpression, HUVECs were transfected with either Empty vector lentivirus or Flag‐Rictor lentivirus (Genechem) following the manufacturer's instruction.

Feng D., Gui Z., Xu Z., Zhang J., Ni B., Wang Z., Liu J., Fei S., Chen H., Sun L., Gu M, & Tan R. (2024). Rictor/mTORC2 signalling contributes to renal vascular endothelial‐to‐mesenchymal transition and renal allograft interstitial fibrosis by regulating BNIP3‐mediated mitophagy. Clinical and Translational Medicine, 14(5), e1686.

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Isolation and Culture of EC and HUVEC Cells

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Human EC cell lines (Ishikawa and RL95-2) were obtained from American Type Culture Collection (ATCC, Manassas, VA). Human umbilical vascular endothelial cells (HUVECs) were obtained from Key-GEN Biotech (Nanjing, China). Ishikawa and RL95-2 cells were grown in DMEM/F12 (Gibco, Auckland, NZ) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA). HUVECs were cultured in RPMI1640 (Gibco) supplemented with 10% fetal bovine serum. Cells were cultured in a 5% CO₂ humidified incubator at 37°C. For hypoxia experiments, cells were grown in an in vitro hypoxic (<0.1% O₂) container system (BD Diagnostics, Sparks, MD). Conditioned medium (CM), protein and RNA were collected immediately when cells were removed from hypoxic container.

Liao Y., Lu W., Che Q., Yang T., Qiu H., Zhang H., He X., Wang J., Qiu M., Zou Y., Gu W, & Wan X. (2014). SHARP1 Suppresses Angiogenesis of Endometrial Cancer by Decreasing Hypoxia-Inducible Factor-1α Level. PLoS ONE, 9(6), e99907.

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Cell Line Characterization and Transfection

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Human RCC cell lines 786-O and OS-RC-2 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human umbilical vascular endothelial cells (HUVECs) were obtained from Key-GEN biotech (Nanjing, China). 786-O, OS-RC-2 and HUVEC cells were cultured in RPMI1640 medium supplemented with 10% fetal calf serum (Invitrogen, Shanghai, China). Cells were in a 37 °C humidified incubator with 5% CO₂. The CHIP overexpression plasmids were obtained from Dr. Shouyu Wang (Nanjing Medical University, Jiangsu, China). non-specific control siRNA or CHIP siRNA was purchased from GenePharma biotech (Shanghai, China). Transfection of the plasmids into the renal carcinoma cells were carried out using Lipofectamine 2000 transfection reagent (Invitrogen, Shanghai, China) following the manufacturer’s protocol. Transfection of the non-specific control siRNA or CHIP siRNA were carried out using siLentFect Lipid Reagent (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions.

Sun C., Li H.L., Chen H.R., Shi M.L., Liu Q.H., Pan Z.Q., Bai J, & Zheng J.N. (2015). Decreased expression of CHIP leads to increased angiogenesis via VEGF-VEGFR2 pathway and poor prognosis in human renal cell carcinoma. Scientific Reports, 5, 9774.

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HUVEC Culture Optimization Protocol

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HUVECs (KeyGen, China) were cultured in RPMI.1640 medium (KeyGen BioTECH, China) supplemented with 10% Fetal Bovine Serum (Gibco, Life technologies, US), 80 U/ml penicillin, and 0.08 mg/ml streptomycin as recommended by the supplier. They were incubated at 37 °C in humidified atmosphere containing 5% CO₂ and 95% air. After reaching 80% confluence, cells were passaged by 0.25 wt.% trypsin-EDTA solution (Gibco, US). Cells at passages 3 to 10 were used for subsequent experiments.

Jin S., Qi X., Zhang B., Sun Z., Zhang B., Yang H., Wang T., Zheng B., Wang X., Shi Q., Chen M., Ren L., Yang K, & Zhong H. (2017). Evaluation of promoting effect of a novel Cu-bearing metal stent on endothelialization process from in vitro and in vivo studies. Scientific Reports, 7, 17394.

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