Endothelial cells form capillary-like structures in response to angiogenic signals found in conditioned media (DeCicco-Skinner et al., 2014 (link)). To confirm endothelial morphology remained unchanged by RPMI media, tube formation assay was performed essentially as described previously (DeCicco-Skinner et al., 2014 (link)). In short, HPMECs were cultured for 4 days in either endothelial cell growth medium (Sciencell) or RPMI (Gibco) with 5% FBS (Sigma), 1% penicillin-streptomycin (Lonza) and either 7 mM glucose or 12 mM glucose (Gibco). After 4 days of culture, a Corning Costar TC-treated 96-well plate (Sigma) was coated with Cultrex Basement Membrane Extract, Type 2 (BME) (Sigma). Once BME set, cells were seeded at a density of 1.5 × 103 cells/mL in respective media. Tubes formed within 2–4 hr and were imaged using an Eclipse Ts2 inverted microscope (Nikon Instruments, Amsterdam, North Holland, the Netherlands).
Endothelial cell growth medium
Manufactured by ScienCell
Sourced in United States
Endothelial cell growth medium is a specialized cell culture medium designed to support the growth and maintenance of endothelial cells in vitro. It contains a balanced combination of essential nutrients, growth factors, and other components required for the optimal proliferation and differentiation of endothelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (4 mentions)
Hpmec, by ScienCell (4 mentions)
Penicillin streptomycin, by Lonza (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Ncl h441 cells, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Nci h441 cells, by American Type Culture Collection (2 mentions)
Hcaec, by ScienCell (2 mentions)
Huvecs, by ScienCell (2 mentions)
Huvecs, by American Type Culture Collection (2 mentions)
Eclipse ts2 inverted microscope, by Nikon (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Egm 2mv medium, by Lonza (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Tns1 sirna, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Huvecs, by Lonza (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
19 protocols using endothelial cell growth medium
1
Endothelial Tube Formation Assay
2
Cultivation of Alveolar Cell Lines and Primary Endothelial Cells
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Human epithelial NCl-H441 cells (which express alveolar type II cell markers [Ren et al., 2016 (link)]) were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in Roswell Park Memorial Institute medium (RPMI) (Gibco, Grand Island, NY) with 10% fetal bovine serum (FBS) (Sigma, St Louis, MO) and 1% penicillin-streptomycin (Lonza, Basel, Switzerland). NC1-H441 cells were used at passages 3–8. Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from Sciencell (Carlsbad, CA) and cultured in endothelial cell growth medium (Sciencell) in fibronectin coated tissue culture flasks (2 μg/cm2, Sigma). HPMEC cells were used at passages 3–7, as at higher passages these cells can demonstrate senescence (Shen et al., 1995 (link)). Madin-Darby canine kidney (MDCK) cells were obtained from ATCC and cultured in Dulbecco modified Eagle medium (Gibco) between passages 20 and 50. All cell lines were maintained in a humidified 37°C incubator with 95% O2 and 5% CO2. Each cell line has no mycoplasma contamination to report.
3
Endothelial and Fibroblast Cell Isolation
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The use of embryos for cell culture was approved by the University of Queensland animal ethics committee (SCMB/002/18). Seventeen-day-old chicken and twenty-one-day-old Pekin duck embryonated eggs were purchased from Darling Downs Hatchery (Queensland, Australia). Primary aortic endothelial cells were cultured from the aortic arches of chicken and duck embryos as described previously using EGM-2MV medium (Lonza, Basel, Switzerland) with 10% FCS (Gibco, Waltham, MA, USA) [7 (link),8 (link)]. Primary chicken and duck embryo fibroblasts were cultured as described previously [9 (link),10 (link)]. Madin–Darby Canine Kidney (MDCK) cells were obtained from American Type Culture Collection (ATCC), and cultured in DMEM (Gibco, Waltham, MA, USA) supplemented with 10% FCS (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (Gibco, Waltham, MA, USA). NCI-H441 cells (human lung epithelial cells) were obtained from ATCC and cultured in RPMI (Gibco, Waltham, MA, USA) medium supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco). Primary human pulmonary microvascular endothelial cells were obtained from Sciencell (Carlsbad, CA, USA) and cultured in an endothelial cell growth medium (Sciencell, Carlsbad, CA, USA). All cell lines of mammalian origin were incubated at 37 °C 5% CO2 whilst all avian cells were cultured at 40 °C 5% CO2 unless otherwise stated.
4
Cell Culture and Transfection Protocol
5
Phosphate-induced Calcification in HUVECs
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HUVECs were obtained from Lonza Group, Ltd. (Basel, Switzerland) and cultured with Endothelial Cell Growth Medium (ScienCell Research Laboratories Carlsbad, CA, USA) in a 37 °C incubator with 5% CO2. An appropriate amounts of Na2HPO4/NaH2PO4 buffer (1 M; pH7.4) was added into the cell culture medium to achieve different phosphorus concentrations (1.0 and 3.0 mM) with or without an inhibitor of Ca-Pi crystal formation, phosphonoformic acid (PFA, 1.0mM; Merck KGaA), as previously described (Di Marco et al., 2008 (link); Peng et al., 2011 (link); Zhou et al., 2016 (link)). Cells were treated with different Pi concentrations in the presence or absence of PFA for 24 h. ERK inhibitor U0126 (100 ng/mL, Thermo Fisher Scientific Inc, Waltham, MA, USA) was used to treat cells for 24 h when necessary, and DMSO was used as a vehicle control.
6
HCAEC Cell Culture Protocol
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HCAECs were purchased from ScienCell Research Laboratories (San Diego, CA, USA). Cells were grown in endothelial cell growth medium (ScienCell Research Laboratories). In these experiments, cells were seeded in 0.5 ml complete medium in 24-well plates. The control cells were the vehicle cells, which were created using HCAECs dissolved in dimethyl sulfoxide.
7
Cell Culture Protocols for NCI-H441, HPMEC, and MDCK
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NCI-H441 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in RPMI medium (Gibco, Grand Island, NE) with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco). NCI-H441 cells were used between passages 2 and 14. Primary human pulmonary microvascular endothelial cells (HPMECs) were obtained from Sciencell (Carlsbad, CA) and cultured in endothelial cell growth medium (Sciencell). HPMECs were used at passages 2 to 7. Madin-Darby canine kidney (MDCK) cells were obtained from ATCC and kept in Dulbecco modified Eagle medium (DMEM; Gibco) with 10% FBS and 1% penicillin-streptomycin. MDCK cells were used between passages 20 and 50. All cell lines were cultured using a humidified 37°C incubator with 95% O2 and 5% CO2.
8
Engineered Biomaterials for Tissue Regeneration
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Poly(dimethylsiloxane) (PDMS) pre-polymer (Sylgard 184) and a curing agent were purchased from Dow Corning (Midland, MI, USA). Tris-HCl was purchased from Biosesang (Seongnam, Korea). Dopamine hydrochloride and Alizarin Red S were obtained from Sigma-Aldrich (St. Louis, MO, USA). MSCs, HUVECs, MSC growth medium (MSCM), endothelial cell growth medium (ECM), endothelial cell growth supplements (ECGS), MSC growth supplements (MSCGS), fetal bovine serum (FBS), penicillin/streptomycin (P/S) solution, trypsin-EDTA (T/E) solution, Dulbecco’s phosphate-buffered saline (DPBS), MSC chondrogenic differentiation medium (MCDM), MSC chondrogenic differentiation supplement (MCDS), MSC osteogenic differentiation medium (MODM), and MSC osteogenic differentiation supplement A and B (MODS-A and MODS-B) were purchased from ScienCell (Carlsbad, CA, USA). Transforming growth factor-β3 (TGF-β3) and live/dead viability/cytotoxicity kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Safranin O was obtained from Polyscience (Warrington, PA, USA). Norland optical adhesive63 (NOA63) was purchased from Norland Products (Jamesburg, NJ, USA). Polystyrene (PS) sheets and poly(ethylene terephthalate) (PET) film were obtained from Goodfellow (Pittsburgh, PA, USA).
9
Transfection of Trophoblast and Endothelial Cells
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Human trophoblastic cell lines (HTR-8/SVneo, JAR, BeWo, and JEG3) and human umbilical vein endothelial cells (HUVECs) were obtained from the Shanghai Institute of Cell Biology (Shanghai, China). HTR-8/SVneo and JAR cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (#E600028; Sangon Biotech, Shanghai, China) supplemented with 10% heat-inactivated fetal bovine serum (#E600001; Sangon Biotech), and HUVEC cells were maintained in endothelial cell growth medium (#1001; ScienCell, USA). All cells were kept at 37 °C in a humidified incubator with 5% carbon dioxide.
Plasmid vectors (plasmid cloning DNA [pcDNA]-GASAL1 and pcDNA-SRSF1) were constructed by GenScript Biotech Corp. (Nanjing, China). The short interfering RNA against GASAL1 (GASAL1 siRNA), SRSF1 (SRSF1 siRNA), and negative control siRNAs were designed, synthesized, and validated by Thermo Fisher Scientific (Waltham, MA, USA). HTR-8/SVneo and JAR cells were seeded in six-well plates at a density of 2 × 105 cells/ml. On reaching about 70% confluence, the cell transfection was performed using Lipofectamine 2000 transfection reagent (#11668019; Invitrogen, Carlsbad, CA, USA) under the suggestion of the manufacturer. Twenty-four hours after transfection with the siRNAs and plasmid vectors, the cells were harvested for further experiments.
Plasmid vectors (plasmid cloning DNA [pcDNA]-GASAL1 and pcDNA-SRSF1) were constructed by GenScript Biotech Corp. (Nanjing, China). The short interfering RNA against GASAL1 (GASAL1 siRNA), SRSF1 (SRSF1 siRNA), and negative control siRNAs were designed, synthesized, and validated by Thermo Fisher Scientific (Waltham, MA, USA). HTR-8/SVneo and JAR cells were seeded in six-well plates at a density of 2 × 105 cells/ml. On reaching about 70% confluence, the cell transfection was performed using Lipofectamine 2000 transfection reagent (#11668019; Invitrogen, Carlsbad, CA, USA) under the suggestion of the manufacturer. Twenty-four hours after transfection with the siRNAs and plasmid vectors, the cells were harvested for further experiments.
10
Cell Culture of A549, HPMEC, and RPTEC
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A549 cells were obtained from the Center for Biological Signaling Studies (BIOSS) Toolbox, University of Freiburg (Germany) and cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin/streptomycin (P/S) (100 U/mL; PAN-Biotech GmbH, Aidenbach, Germany). HPMEC were procured from ScienCell Research Laboratories (Carlsbad, CA, USA) and grown in endothelial-cell growth medium (ScienCell) supplemented with 5% fetal bovine serum and 1% P/S. RPTEC were purchased from Lonza Group (Basel, Switzerland) and were grown in renal-cell growth media (REBM basal medium, Lonza). All cells were maintained in a humidified incubator at 37°C with 5% CO2.
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