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23 protocols using huvecs

1

Cultivation of Human Umbilical Vein Endothelial Cells

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Human umbilical vein endothelial cells (HUVECs) were purchased from Allcells, China. HUVECs were grown in complete culture media for HUVECs (Allcells, China) in a humidified 5%/95% CO2/air incubator at 37 °C. Fluorescence-activated cell sorting (FACS) analysis showed that almost all of the cells (>96%) take up high amount of acetylated LDL and are positive for the presence of CD31, CD45, CD62, and von Willebrand factor (vWF). Cells (passages 3–5) were plated on to glass slides or 6-well plates at a density of 1 × 105 cells/cm2 and cultured for 3–5 days until they attained confluence.
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2

HUVEC Culture in Normal and High Glucose

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The HUVECs were purchased from ALLCELLS (Shanghai, China). The HUVECs from passages 4 to 7 were used in the experiment and were cultured in endothelial completed medium (ALLCELLS, Shanghai, China) at 37 °C in 5% CO2. The HUVECs were cultured in normal glucose medium (NG; 5.5 mmol/L of D-glucose) or high-glucose medium (HG; 33.3 mmol/L of D-glucose) for 48 h, and the L-glucose (27.8 mmol/L) contained in NG was used as the osmotic pressure control.
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3

Isolation and Culture of DPSCs and HUVECs

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The present study was approved by the Ethics Committee of Sun Yat-sen University [No. ERC-(2017)-34]. Human DPSCs were obtained from healthy pulp tissues harvested from wisdom teeth without caries, which were extracted from 10 donors (both male and female, 22-36 years old) between September, 2018 and March, 2019 at the Hospital of Stomatology, Sun Yat-sen University, Guangzhou, China. Dental pulp tissues were minced and digested for the isolation of DPSCs as previously described (22 (link)). DPSCs were cultured in α-MEM with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (MilliporeSigma). HUVECs were purchased from AllCells, LLC and expanded in complete endothelial cell growth medium (EGM; AllCells Biotech Shanghai Co., Ltd.). For the exosome co-culture experiment, 10 µg/ml exosomes were added to the culture medium of HUVECs. All cells were maintained at 37°C in an incubator contained 5% CO2.
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4

Isolation and Characterization of Human Pancreatic Stellate Cells

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Aspc-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and HUVECs were obtained from ALLCELLS (Emeryville, California, USA). In our experiments, only early-passage cells were used. Human PSCs were extracted from normal pancreatic tissues that had been isolated from donor patients who underwent liver transplantation at the Department of Hepatobiliary Surgery of the First Affiliated Hospital of Xi'an Jiaotong University. The isolation and PSC culture methods were as described in previous studies [8 (link), 25 (link), 26 (link)]. Morphological examination and oil red O staining of intracellular fat droplets were used to assess the purity of the PSCs, and α-smooth muscle actin expression in PSCs was assessed by immunofluorescence (see Supplementary Fig. S1). The PSCs used in this study were obtained from several patients. The written consent from patient's family was obtained, and the study protocol and consent forms were approved by the relevant ethical committee of the First Affiliated Hospital of Xi'an Jiaotong University, China.
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5

Culturing HCC and Endothelial Cells

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Human HCC cell lines, HCCL-M3 and MHCC-97L (established at our institute) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen), 100
IU/mL penicillin G and 100 mg/mL streptomycin sulfate (Sigma-Aldrich, St. Louis, MO, USA) in a humidified atmosphere of 5% CO2 at 37°C.
TECs and normal ECs (NECs) were isolated from surgical HCC specimens and the surrounding normal liver tissues, as previously described (29 (link)). Primary human umbilical vein endothelial cells (HUVECs; AllCells, Shanghai, China) were cultured in endothelial cell growth medium-2 (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum and were used within 2-10 passages.
Conditioned media (CM) for ECs was generated according to our previous study (30 (link)).
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6

Cell Culture and Transfection Protocols

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Human embryonic kidney 293 T (HEK293T) cells were purchased from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. Neuro-2A (N2A) cells were provided by Dr. Ardem Patapoutian at the Scripps Research Institute and cultured in Modified Eagle Medium (MEM) containing 10% FBS, non-essential amino acids, 1 mM sodium pyruvate, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. Human umbilical vein endothelial cells (HUVECs) were purchased from Allcells (Shanghai, China) and cultured using EGM-2 growth medium supplemented with EGM-2 bullet kit (Lonza) in the plates coated with 50 μg ml−1 collagen-I (Sigma). HUVECs were used for the experiments for up to 8 passages. The cells were transfected using polyethylenimine (PEI) (Polysciences) or Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
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7

Silencing SVIL in Liver Cancer Cells

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Human umbilical vein endothelial cells (HUVECs) were purchased from AllCells LLC (cat. no. H-001F-C) and cultured with HUVEC medium (cat. no. H-004; AllCells LLC) in a 0.25% gelatin-coated culture flask. HepG2, Bel7405 and MHCC-97H liver cancer cell lines were cultured in DMEM medium (cat. no. C11995; Gibco; Thermo Fisher Scientific, Inc.) and provided by Professor ZY Tang (Liver Cancer Institute, Fudan University, Shanghai, China), which were used in previous studies (22 (link),26 (link)). These cell lines were characterized by DNA fingerprinting and isozyme detection. All cell lines used in the present study were regularly authenticated via morphologic observation and tested for the absence of mycoplasma contamination. Samples were last tested for mycoplasma in March 2017.
Cells were transfected with SVIL Stealth siRNA: E4 double stranded (ds)RNA, E5 dsRNA, E11 dsRNA and negative control dsRNA (all Invitrogen; Thermo Fisher Scientific, Inc.; all 40 nM) using Lipofectamine® RNAi-MAX (Invitrogen; Thermo Fisher Scientific, Inc.). The dsRNA targeting sequences were as follows: E4 dsRNA, 5′-CUCACUUUGAAUGUAGAGAACCAUC-3′; E5 dsRNA, 5′-UUCUGCUGAAGUUAUAGGUUGGGUU-3′; E11 dsRNA, 5′-AGCAUAUUUAGAUUCCUUAUGGCUG-3′.
HepG2 cells were treated with or without 50 µg/l recombinant human VEGF (Novus Biologicals, LLC) at the indicated time-points.
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8

Endothelial Cell Culture and PM2.5 Treatment

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HUVECs and HMEC-1 human microvascular endothelial cells were obtained from AllCells, LLC (H-001F; Shanghai, China). Cells were cultured in RPMI 1640 medium with 20% FBS, 60 µg/ml of endothelial cell growth supplement (BD Biosciences, San Jose, CA, USA) and 100 U/ml of penicillin with 100 µg/ml of streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified atmosphere with 5% CO2 at 37°C.
PM2.5 was resuspended in PBS and the solutions were stored at −20°C until use. ECs were treated with various concentrations of PM2.5 to select the optimal concentration and time.
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9

Comparative Cell Proliferation and Migration Assays

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For the HUVEC proliferation, apoptosis, and cell cycle analysis assays, HUVECs (Sciencell Research Laboratories, Carlsbad, CA, USA, #8000) were cultured in ECM medium (ScienCell, #1001) containing 5% FBS (ScienCell, #0025), 1% penicillin/streptomycin (GIBCO, Invitrogen Inc., Carlsbad, CA, USA, #15140–122) and 1% ECGS (ScienCell, #1052). For HUVEC Western blot, migration inhibition, and scratch assays, HUVECs (AllCells, Alameda, CA, USA) were maintained in HUVEC medium (Allcells, #H004B) with 10% FBS (Allcells, #H005) and HUVEC growth factors (Allcells, #H005). HUVECs at passages 4–8 were used in the experiments. The human non-small cell lung cancer cell line PC9 was purchased from Riken BioResource Research Center (Ibaraki, Japan, #RCB4455). The mouse lung cancer cell line 3LL was purchased from JCRB Cell Bank of the National Institutes of Biomedical Innovation, Health, and Nutrition (Tokyo, Japan, #JCRB-1348). The mouse colorectal cell line CT26 was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA, #CRL-2638). The PC9, 3LL, and CT26 cells were cultured in RPMI-1640 medium (Gibco, #22400–089) with 10% FBS (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA, #SV30087.03), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, #15240–062).
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10

HUVEC Angiogenesis Model with bFGF

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HUVECs were from AllCells (Shanghai, China). They were put in an RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, USA), 60 μg/ml of endothelial cell growth supplement (ThermoFisher Scientific, WA, USA) and 100 U/ml of penicillin with 100 μg/ml of streptomycin (Gibco). The cells were incubated in an incubator at 37 °C, with 5% CO2.
To simulate the in vitro CRNV models, HUVECs were treated with 5, 10 or 20 ng/ml essential fibroblast growth factor (b-FGF) for 24 h.
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