Human umbilical vein endothelial cells (HUVECs) (PromoCell GmbH, Heidelberg, Germany) were used to test the effect of the bacteria on vascular properties. After thawing, the frozen HUVECs were expanded in low-serum endothelial cell growth medium (PromoCell) at 37°C with 5% CO2 in a humidifying incubator and used at passages p3 to p6 (30 (link), 44 (link)). Cells were grown to 80 to 90% confluence before being transferred to transparent polyethylene terephthalate (PET) Transwell supports (0.4 μm pore size, Greiner Bio-One, Austria), to a plastic well plate (Corning), to a glass-bottom well plate (Cellvis, Mountain View, CA), and to the insert chip (30 (link)). Before seeding, the uncoated substrates were treated with entactin-collagen IV-laminin (ECL) cell attachment matrix (Merck) diluted in Dulbecco’s modified Eagle’s medium (DMEM) (10 μg/cm2) for 1 h in the incubator. Then, the HUVECs, harvested with trypsin/EDTA solution (Biological Industries), were seeded inside the culture platforms at a density of 250,000 cells/cm2 and grown for 48 h. Then, bacteria were added and their effect on cell behavior was tested after 1 h, 4 h, and 24 h.
Low serum endothelial cell growth medium
Manufactured by PromoCell
Low-serum endothelial cell growth medium is a specialized cell culture medium designed to support the growth and maintenance of endothelial cells. This medium provides essential nutrients and growth factors required for the proliferation and survival of endothelial cells while utilizing a reduced serum concentration.
Automatically generated - may contain errors
Lab products found in correlation
Entactin collagen 4 laminin ecl cell attachment matrix, by Merck Group (3 mentions)
Huvecs, by PromoCell (3 mentions)
Glass bottom well plate, by Cellvis (2 mentions)
Huvecs, by Sartorius (1 mentions)
Trypsin edta solution, by Sartorius (1 mentions)
Vegf165, by Thermo Fisher Scientific (1 mentions)
Sdf 1α, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
S8apo, by Leica (1 mentions)
Gentamycin, by Merck Group (1 mentions)
Low serum growth supplement, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
C 12200, by PromoCell (1 mentions)
Detachkit, by PromoCell (1 mentions)
6 protocols using low serum endothelial cell growth medium
1
Endothelial Cell Culture and Bacterial Interaction
2
HUVEC Barrier Function Assay
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Human umbilical vein endothelial cells (HUVECs; PromoCell GmbH, Heidelberg, Germany) were used to test the impact on cell-barrier properties. HUVECs were cultured in low-serum endothelial cell growth medium (PromoCell) at 37°C with 5% CO2 in a humidifying incubator and used at passage p4–p6. Cells were cultured on transparent polyethylene terephthalate Transwell supports (0.4-μm pore size; Greiner Bio-One, Kremsmünster, Austria), coated with Entactin-Collagen IV-Laminin (E-C-L) Cell Attachment Matrix (Merck Millipore, Burlington, MA) diluted in Dulbecco's modified Eagle's medium (10 μg/cm2) for 1 h before seeding, at a density of 40,000 cells/cm2, and grown for 3 days.
3
Heparin-RGD Hydrogel Encapsulation of Endothelial Cells and Growth Factors
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Hydrogels were prepared as described previously19 (link). Briefly, 10 mg of heparin-maleimide conjugate (MW 15000) was dissolved in 107 μl serum-free medium. Subsequently, the heparin solution was added to 0.79 mg cRGD (MW 594, Peptides International, Louisville, KY, USA) at a final concentration of 3 mM and incubated at room temperature for 30 minutes. Next, heparin-RGD solution was mixed with cells and human recombinant growth factors: VEGF165, bFGF, SDF1α (PeproTech, USA). The final concentration of each growth factor was 5 μg/ml. 10 mg of PEG-MMP conjugate (MW 15920) was dissolved in 372 μl of ice-cold serum-free medium. Heparin-RGD-cell-growth factor solution was mixed gently with PEG-MMP solution in ration 1:1 and gel formation occurred within several seconds. The final endothelial cell concentration was 6 × 106/ml, the concentration of supporting cells varied between 5–25% of the endothelial cells. Upon polymerization, gels were immediately immersed in low-serum Endothelial Cell Growth Medium (PromoCell) with 1% penicillin/streptomycin and cultured as floating droplets. 70% of the medium was exchanged every other day. The size of the hydrogel droplets was monitored daily by stereo microscope (Leica S8APO).
4
Isolation and Culture of Primary Human Cell Types
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Primary human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords as described elsewhere24 (link). Cells were maintained in low-serum Endothelial Cell Growth Medium (PromoCell) with 1% penicillin/streptomycin and utilized within the 3–6th passage.
Primary human aortic smooth muscle cells (SMCs) and primary human dermal fibroblasts (HDFs) were purchased from ATCC. SMCs were maintained in low-serum Smooth Muscle Cell Growth Medium 2 (PromoCell) with 1% penicillin/streptomycin and utilized within the 5–6th passage. HDFs were maintained in low-serum Medium 106 supplemented with Low Serum Growth Supplement (Gibco) with 1% penicillin/streptomycin and utilized within the 7–8th passage.
Human bone marrow mesenchymal stem cells (MSCs) were isolated from healthy donors (male/female, Caucasians 21–35 years of age) after obtaining their informed consent. The study was approved by the institutional review board of the Medical Faculty at the University Hospital Dresden. MSCs were expanded, characterized and cultured as described previously50 . MSCs were maintained in DMEM (Gibco) with 10% FCS (Biochrom) and 1% penicillin/streptomycin and utilized within the 1–2nd passage.
HepG2 human hepatocarcinoma cells were cultured in RPMI medium (Gibco) supplemented with 10% heat inactivated bovine serum and 50 μg/ml gentamycin (Sigma-Aldrich).
Primary human aortic smooth muscle cells (SMCs) and primary human dermal fibroblasts (HDFs) were purchased from ATCC. SMCs were maintained in low-serum Smooth Muscle Cell Growth Medium 2 (PromoCell) with 1% penicillin/streptomycin and utilized within the 5–6th passage. HDFs were maintained in low-serum Medium 106 supplemented with Low Serum Growth Supplement (Gibco) with 1% penicillin/streptomycin and utilized within the 7–8th passage.
Human bone marrow mesenchymal stem cells (MSCs) were isolated from healthy donors (male/female, Caucasians 21–35 years of age) after obtaining their informed consent. The study was approved by the institutional review board of the Medical Faculty at the University Hospital Dresden. MSCs were expanded, characterized and cultured as described previously50 . MSCs were maintained in DMEM (Gibco) with 10% FCS (Biochrom) and 1% penicillin/streptomycin and utilized within the 1–2nd passage.
HepG2 human hepatocarcinoma cells were cultured in RPMI medium (Gibco) supplemented with 10% heat inactivated bovine serum and 50 μg/ml gentamycin (Sigma-Aldrich).
5
3D Vascularized Liver Spheroid Model
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7 day old HepG2 spheroids were extracted from the 0.2 kPa hydrogel containing 3 mM cRGD, w/o growth factors by mechanical dispersion and incorporated together with the endothelial cell suspension in a fresh 0.2 kPa hydrogel. The 3D cultures were maintained in low-serum Endothelial Cell Growth Medium (PromoCell) with 1% penicillin/streptomycin. 70% of the medium was exchanged every other day. The spheroids and endothelial cell co-culture was terminated on day 3, when the capillaries were fully developed, and fixed with 4% PFA. Hydrogels were embedded in OCT and subjected to cryosectioning, followed with immunostaining with anti-CD31 antibody and phalloidin as described above. Samples were imaged with Leica SP5 confocal microscope (20×, 0.70; 40×, 1.25; 63×, 1.40). 200 μm thick z-stacks were acquired. Three-dimensional reconstruction was performed using Volocity software.
6
Investigating Viral Protein Impact on Endothelial Cells
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HUVECs (C-12200, PromoCell GmbH, Heidelberg, Germany, tested negative for mycoplasma contamination) were used to test each viral protein’s impact on vascular properties. After thawing, the HUVECs were expanded in low-serum endothelial cell growth medium (PromoCell) at 37°C with 5% CO2 in a humidifying incubator, and used at passage p4–p6. Cells were grown to 80–90% confluence before being transferred to transparent polyethylene terephthalate Transwell supports (0.4 µm pore size, Greiner Bio-One, Austria) or a glass-bottom well plate (Cellvis, Mountain View, CA). Before seeding, the uncoated substrates were treated with Entactin-Collagen IV-Laminin (ECL) Cell Attachment Matrix (Merck) diluted in DMEM (10 µg/cm2) for 1 hr in the incubator. Then, the HUVECs were harvested using a DetachKit (PromoCell), were seeded inside the culture platforms at a density of 250,000 cells/cm2, and grown for 3 days. Then viral infection with the different plasmids was performed and its impact on cell behavior was tested 3 days later.
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