The HUVECs (Cyagen, Suzhou, China) was used in these experiments to test the viability, adhesion, and proliferation of HUVECs on the prepared samples. Briefly, HUVECs (5 × 104 cells/cm2) were seeded onto different samples and cultured in endothelial cell medium (ECM, Sciencell, Carlsbad, CA, USA) supplemented with fetal bovine serum (10%, Gibco, Suzhou, China) and Penicillin-Streptomycin (1%, Gibco, Suzhou, China). After 1, 2, and 3 days of incubation, the Live/Dead kit (Thermo Fisher, Suzhou, China) and a CCK-8 quantification assay (Beyotime, Shanghai, China) were used to study the adhesion, viability, and proliferation of HUVECs [19 (link)].
Huvecs
Manufactured by Cyagen
Sourced in China, United States
HUVECs (Human Umbilical Vein Endothelial Cells) are primary endothelial cells derived from the human umbilical vein. They serve as a model for the study of endothelial cell biology and function.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Exosome free serum, by System Biosciences (3 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
Huvecs, by ScienCell (2 mentions)
Endothelial cell medium (ecm), by ScienCell (2 mentions)
M csf, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Mscs complete medium, by Cyagen (1 mentions)
Bm mscs, by Cyagen (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Neofx transfection agent, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Boster Bio (1 mentions)
Cell counting kit 8 cck 8, by Boster Bio (1 mentions)
Bmscs, by Thermo Fisher Scientific (1 mentions)
293t cell, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
Ebm 2, by Lonza (1 mentions)
13 protocols using huvecs
1
Evaluating HUVEC Viability and Adhesion
2
Culturing BM-MSCs and HUVECs
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BM-MSCs of Sprague-Dawley (SD) rat were purchased from Cyagen Biosciences (Guangzhou, China) and human umbilical vein endothelial cells (HUVECs) were purchased from Cobioer Biotechnology (Nanjing, China). BM-MSCs were cultured in MSCs complete medium (Cyagen Biosciences) and HUVECs were cultured in endothelial cell (EC) complete medium (Cyagen Biosciences). All cells were maintained at 37°C in a 5% CO2 incubator.
3
Culturing Human Umbilical Vein Endothelial Cells
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HUVECs were purchased from Cyagen Biosciences Inc. The cells were cultured in endothelial medium from the EGM-2 Bullet Kit (Lonza, Switzerland) supplemented with 10% foetal bovine serum (FBS) (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) in a 5% CO2 incubator at 37 °C. The cells used for subsequent experiments were between passages three and six.
4
Culturing Human Umbilical Vein Endothelial Cells
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Human umbilical vein endothelial cells (HUVECs) were purchased from Cyagen Biotechnology (United States), and cultured with Dulbecco’s Modified Eagle’s Medium (DMEM high glucose, Gibco, United States) containing 10% fetal bovine serum (FBS, Biological Industries, United States) and 1% penicillin-streptomycin (Gibco, United States) at 37°C in a humidified and 5% CO2 incubator.
5
Hypoxia and Serum Starvation in HUVECs
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Human umbilical vein endothelial cells (HUVECs) were purchased from Cyagen Biosciences Inc., and grown in standard endothelial cell growth medium (Cell Applications, Inc.) with 10% fetal bovine serum (Boster Biological Technology). To mimic endothelial cells under ischemic conditions as a model for PAD, HUVECs were subjected to hypoxia (3% oxygen; BioSpherix Medical) and serum starvation (HSS). The reason why the present study used combined HSS to mimic in vivo ischemia is that there is no oxygen or nutrition supply to the ischemic tissue without a blood supply. In vitro transfection of miRNA inhibitors or mimics at a concentration of 10 nM was used to knockdown miR-210 expression in HUVECs, as previously described (17 (link)). Briefly, a reverse transfection protocol using neofx transfection agent (Ambion; Thermo Fisher Scientific, Inc.) was used to transfect the miR-210 mimic (assay ID no. MH10516, Thermo Fisher Scientific, Inc.), miR-210 inhibitor (assay ID no. MH10516; Thermo Fisher Scientific, Inc.) or mirVanaTM negative control miRNA (cat no. 4464060; Thermo Fisher Scientific, Inc.) into HUVECs for 48 h at 37˚C in cell culture incubator, the concentration used for miR mimic or inhibitor transfection was 10 nM.
6
Cell Proliferation Assay of BMSC, HUVEC, 293T
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Sprague-Dawley rat BMSCs, HUVECs, and 293T cells were purchased from Cyagen Biosciences Inc. (Suzhou, China). BMSCs and 293T cells were cultured in DMEM/F12 medium (Gibco, NY, USA) containing 10% foetal bovine serum (FBS, Gibco, NY, USA) and 1% of an antibiotic-antimycotic solution (Sigma-Aldrich, NY, USA). HUVECs were cultured in endothelial basal medium (EBM-2, Lonza, Switzerland) containing endothelial growth supplement (EGM-2). All the cells were maintained at 37 °C in a humidified incubator with 5% CO2.
Cell proliferation was assessed with a Cell Counting Kit-8 (CCK-8, Boster, China). The proliferation of seed cells was measured after 1, 3, and 5 days following the manufacturer’s instructions.
Cell proliferation was assessed with a Cell Counting Kit-8 (CCK-8, Boster, China). The proliferation of seed cells was measured after 1, 3, and 5 days following the manufacturer’s instructions.
7
Endothelial-Mesenchymal Transition Model
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Human umbilical vein endothelial cells (HUVECs) were purchased from Cyagen (#HUVEC-2001). The HUVECs were cultured in a 1% gelatin coated plate in complete endothelial cell medium (ECM, #1001, ScienCell) containing 5% FBS, and endothelial growth factor and were maintained in a humidified chamber with 5% CO2 at 37°C. Human umbilical vein smooth muscle cells (HVSMCs) were obtained from Otwo Biotech (#HTX2305) and maintained in DMEM supplemented with 10% FBS and 1% P/S. To establish the EndMT model, HUVECs were stimulated with 10 ng/ml TGF-β2 (#100-35B, Proteintech) in conditioned medium supplemented with 2.5% FBS for four consecutive days. For DSY application, HUVECs pretreated with TGF-β2 for 2 days were incubated with different doses of DSY for a further 2 days.
8
Osteogenic Co-culture of Endothelial and Mesenchymal Cells
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HUVECs (Cyagen Biosciences Inc., CA, USA) were cultured in Endothelial Growth Medium-2 (EGM-2; Lonza, MA, USA). Before their integration in a 3D co-culture, sGMSCs and HUVECs were collected by trypsin. The osteogenic induction medium in the GS and GHS systems is composed of a combination of EGM-2 and DMEM osteogenic media tailored to match the cell proportions. The induction medium was renewed every 48 h to maintain optimal nutrition for the cellular spheroids.
As a control, sGMSCs and HUVECs were respectively cultured in ultra-low attachment 6-well plates (Corning, NY, USA) at a density of 1×106 cells per well to assemble GS and HUVEC Spheroids (HS). To form GHS with varying ratios, single-cell suspensions of sGMSCs and HUVECs were arranged at a total cellular density of 1×106 cells per well, with GHR of 5:1, 4:1, 3:1, 2:1, and 1:1. These mixed single-cell suspensions were then propagated in ultra-low attachment 6-well plates and subjected to unbroken osteogenic induction for 7–14 days. The morphology of the generated GS and GHS were inspected under a microscope 12–48 h post-seeding, and their diameters were quantified using ImageJ (n = 3).
As a control, sGMSCs and HUVECs were respectively cultured in ultra-low attachment 6-well plates (Corning, NY, USA) at a density of 1×106 cells per well to assemble GS and HUVEC Spheroids (HS). To form GHS with varying ratios, single-cell suspensions of sGMSCs and HUVECs were arranged at a total cellular density of 1×106 cells per well, with GHR of 5:1, 4:1, 3:1, 2:1, and 1:1. These mixed single-cell suspensions were then propagated in ultra-low attachment 6-well plates and subjected to unbroken osteogenic induction for 7–14 days. The morphology of the generated GS and GHS were inspected under a microscope 12–48 h post-seeding, and their diameters were quantified using ImageJ (n = 3).
9
Culturing Human Vascular Cells
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Human umbilical vein endothelial cells (HUVECs) were purchased from Cyagen Biosciences (Guangdong, China) and cultured in endothelial basal medium-2 supplemented with EGM-2 (Lonza, Basel, Switzerland). Human aortic smooth muscle cells (SMCs) were purchased from LIFELINE cell technology (Frederick, MD, USA) and cultured in VascuLife Basal Medium (LIFELINE cell technology, Frederick, MD, USA). GW4869 (Sigma-Aldrich, St. Louis, MO, USA) was added to the expansion medium as needed.
10
Macrophage-Conditioned Medium Modulates HUVEC
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Bone-marrow-derived macrophages (BMDMs, 1 × 106 cells/each well in 6-well plate) were collected from 6 to 8-week-old mice via flushing of the femur and tibia, and were cultured in DMEM (Gibco, New York, United States) medium supplemented with 100 ng/mL M-CSF (Sigma-Aldrich Co. St. Louis, MO) for 5 days. After 36 h of treatment with dapagliflozin, macrophages were subjected to an exposure of 100 ng/mL LPS (Sigma-Aldrich Co. St. Louis, MO) for 12 h, following which they were deemed the conditioned medium (CM) group. Macrophages that were exposed to 100 ng/mL LPS for 12 h without any prior treatment were categorized as Con group macrophages. The resulting CM from these macrophage cells was used to replace the medium for the subsequent incubation of HUVECs (Cyagen Biosciences).
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