To test monocytic adhesion on endothelial cells, isolated monocytes were stimulated with i.v. iron preparations for 3 h at 37°C (3 × 105 monocytes per condition) and then washed with RPMI 1640. Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell media (both from PromoCell) in fibronectin-coated 12-well plates. HUVEC monolayers were washed away with RPMI 1640 before iron-stimulated monocytes were added. After 30 min of incubation, non-adherent monocytes were washed away with RPMI 1640 and the number of adherent monocytes was evaluated by phase contrast microscopy (Biozero BZ-8000, Keyence, Neu-Isenburg, Germany) in 10 microscopic fields per sample.
Endothelial cell media (ecm)
Manufactured by PromoCell
Sourced in Germany
Endothelial cell media is a specialized culture medium designed to support the growth and maintenance of endothelial cells, which are the cells that line the interior of blood vessels and lymphatic vessels. This media provides the necessary nutrients, growth factors, and other components required for the optimal cultivation of endothelial cells in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Biozero bz 8000, by Keyence (1 mentions)
Human umbilical vein endothelial cells huvecs, by PromoCell (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Growth medium supplement mix, by PromoCell (1 mentions)
Hmscs, by Lonza (1 mentions)
Epithelial growth factor (egf), by Merck Group (1 mentions)
Penicillin, by Sartorius (1 mentions)
Human umbilical vein endothelial cells (huvec), by PromoCell (1 mentions)
Streptomycin, by Sartorius (1 mentions)
Insulin, by Merck Group (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Nystatin, by Sartorius (1 mentions)
Exosome depleted fetal bovine serum, by Thermo Fisher Scientific (1 mentions)
N acetylcysteine nac, by Merck Group (1 mentions)
Arpe 19, by Thermo Fisher Scientific (1 mentions)
Arpe 19 human cell line, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle s medium f12, by Thermo Fisher Scientific (1 mentions)
Hdlec, by PromoCell (1 mentions)
5 protocols using endothelial cell media (ecm)
1
Monocyte Adhesion to Endothelial Cells
2
Culturing hMSCs and HUVECs for Research
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hMSCs were purchased from Lonza (Bazel, Switzerland) and cultured in low glucose Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco BRL) and 1% (v/v) penicillin/streptomycin (Gibco BRL) at 37°C with 5% CO2. The cell culture medium was changed every 2 days. In this study, hMSCs within eight passages were used in the experiments. Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and cultured in endothelial cell media (PromoCell) supplemented with Growth Medium SupplementMix (PromoCell) at 37°C with 5% CO2. The medium was changed every 2 days. Cells within four passages were used in the experiments.
3
Culturing HUVEC and Smooth Muscle Cells
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Human umbilical vein endothelial cells (HUVEC) (PromoCell) were cultured in endothelial cell media (PromoCell) supplemented with 100 U/ml Penicillin, 0.1 mg/ml Streptomycin and 12.5 U/ml Nystatin (Biological Industries, Beit Haemek, Israel).
Human umbilical aortic smooth muscle cells (SMC) (Lonza) were cultured in MCDB-131 Medium supplemented with 5% Fetal Bovine Serum, 2 mM L-Glutamine, 100 U/ml Penicillin, 0.1 mg/ml Streptomycin and 12.5 U/ml Nystatin (Biological Industries, Beit Haemek, Israel), 2 ng/ml Insulin (Sigma Aldrich), 0.5 ng/ml Epithelial Growth Factor (Sigma Aldrich), 3 ng/ml Basic-Fibroblasts Growth Factor (bFGF) (PeproTech Inc., USA) and 1 µg/ml Hydrocortisone (Sigma Aldrich). Both cell types as well as the co-culture were cultured at 37°C and 5% CO2 in a humidifier incubator. In both cell types, only passages 4–8 were used for this study.
Human umbilical aortic smooth muscle cells (SMC) (Lonza) were cultured in MCDB-131 Medium supplemented with 5% Fetal Bovine Serum, 2 mM L-Glutamine, 100 U/ml Penicillin, 0.1 mg/ml Streptomycin and 12.5 U/ml Nystatin (Biological Industries, Beit Haemek, Israel), 2 ng/ml Insulin (Sigma Aldrich), 0.5 ng/ml Epithelial Growth Factor (Sigma Aldrich), 3 ng/ml Basic-Fibroblasts Growth Factor (bFGF) (PeproTech Inc., USA) and 1 µg/ml Hydrocortisone (Sigma Aldrich). Both cell types as well as the co-culture were cultured at 37°C and 5% CO2 in a humidifier incubator. In both cell types, only passages 4–8 were used for this study.
4
In vitro ARPE-19 cell oxidative stress
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The arising retinal pigment epithelium (ARPE-19) human cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). ARPE-19 cells were cultured in Dulbecco’s modified Eagle’s medium/F12 (Invitrogen, Carlsbad, CA, USA), as previously described [24 (link)]. Cells were used from 11 to 30 passages. Cells were cultured to 80–90% confluence at a seeding density of 1 × 106 cells/cm2. After 48 h of seeding, cells were treated with 600 µM H2O2 (Scharlau, Barcelona, Spain) and/or 4 mM N-acetylcysteine (NAC; Sigma-Aldrich, St. Louis, MO, USA) for 24 h grown in media supplemented with 1% of exosome-depleted fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA). Cell media and cells were collected and preserved for future experiments.
Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical veins as previously described [36 (link)]. HUVEC were grown in endothelial cell media (PromoCell, Heidelberg, Germany) supplemented with 20% FBS, penicillin/streptomycin, and amphotericin at 37 °C and 5% CO2.
Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical veins as previously described [36 (link)]. HUVEC were grown in endothelial cell media (PromoCell, Heidelberg, Germany) supplemented with 20% FBS, penicillin/streptomycin, and amphotericin at 37 °C and 5% CO2.
5
Isolating and Culturing Endothelial Cells
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Umbilical cords were supplied by Bradford Royal Infirmary under the approval and processing of Ethical Tissue Bradford. HUVEC (Human Umbilical Vein Endothelial Cells) from 5 donors were isolated from umbilical cords in a previously described method (30) . HDLEC (Human Dermal Lymphatic Endothelial Cells) from pooled donors were supplied from PromoCell (Heidelberg, Germany). Cells were incubated in an atmosphere of 95% humidity and 5% CO2 at 37°C. All experiments were performed between the passages of 2-5 for both cell types.
Endothelial cell media (PromoCell) was used containing penicillin/streptomycin (100U/100mg/ml) and fungizone (2.5 µg/ml) (both Life Technologies, Carlsbad, USA). Cells were plated out on 6 well plates (Greiner Bio-One, Stonehouse, UK) pre coated with 10% v/v gelatine solution overnight. Relevant cells were incubated with NF-B inhibitor (IMD-0354) (Merck Millipore, Billerica,USA) 1µM for 1 hour prior to stimulation. The specificity of IMD-0354 at the doses used as previously been documented (31) .
Endothelial cell media (PromoCell) was used containing penicillin/streptomycin (100U/100mg/ml) and fungizone (2.5 µg/ml) (both Life Technologies, Carlsbad, USA). Cells were plated out on 6 well plates (Greiner Bio-One, Stonehouse, UK) pre coated with 10% v/v gelatine solution overnight. Relevant cells were incubated with NF-B inhibitor (IMD-0354) (Merck Millipore, Billerica,USA) 1µM for 1 hour prior to stimulation. The specificity of IMD-0354 at the doses used as previously been documented (31) .
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