Fifty-five de-identified umbilical cords were obtained from the University of Maryland Medical Center Division of Maternal and Fetal Medicine under an IRB exempt protocol due to their de-identified nature. Upon receipt of umbilical cords, HUVECs were harvested as described previously [22 (link)]. HUVECS were maintained at 37°C in a 5% CO2 incubator using Endothelial Basal Media 2 (Lonza, Catalog #CC-3156 & CC-4176, Walkersville, Maryland) containing 2% FBS, 0.04% hydrocortisone, 0.4% hFGF, 0.1% VEGF, 0.1% R3-IGF, 0.1% ascorbic acid, 0.1% hEGF, 0.1% gentamicin-amphotericin-B [GA-1000], and 0.1% heparin. DNA from each cell line was extracted using a Gentra Puregene Cell Kit (Qiagen, Valencia, California) as recommended by the manufacturer. PEAR1 rs12041331 genotype was determined using a TaqMan SNP genotyping assay (Applied Biosystems/Life Technologies, Foster City, California), which resulted in the identification of 28 major allele homozygotes (GG), 25 heterozygotes (GA), and 2 minor allele homozygotes.
Endothelial basal media 2
Manufactured by Lonza
Sourced in United States
Endothelial Basal Media-2 is a basic culture medium designed for the growth and maintenance of endothelial cells in vitro. It provides the necessary nutrients and growth factors to support the specific requirements of endothelial cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Gentra puregene cell kit, by Qiagen (1 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (1 mentions)
Cc 2530, by Lonza (1 mentions)
Cc 3162, by Lonza (1 mentions)
Ebm 2 singlequots, by Lonza (1 mentions)
Pooled human umbilical vein endothelial cells huvecs, by Lonza (1 mentions)
Trypsin edta, by Lonza (1 mentions)
Hepg2 cell, by American Type Culture Collection (1 mentions)
4 protocols using endothelial basal media 2
1
Umbilical Cord Derived HUVEC Protocol
2
Nrf2 Pathway Activation in Cell Lines
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We utilized the human HepG2 cell line (hepatocellular carcinoma) and primary human pulmonary artery endothelial cells (HPAEC) for genomic assays. HepG2 and HPAEC cells are suitable models in the present work because they each have a Nrf2 pathway that responds in a normal manner to Nrf2 activators [33 (link),34 (link)], and do not have reported mutations in Nrf2/KEAP1. The HepG2 cells were cultured and maintained by standard methods, using Opti-MEM medium with 4% fetal bovine serum (FBS) and geneticin/penicillin/streptomycin. HPAEC cells were procured from Lonza (catalog # CC-2530) and cultured in Endothelial Basal Media-2 (Lonza catalog #: CC-3516) supplemented with endothelial growth factors optimized for aortic and pulmonary arterial endothelial cells (Lonza catalog # CC-3162). HPAEC subculturing was limited to six passages in order to prevent senescence and de-differentiation. HPAEC were seeded at a density of 5 × 105 cells per 100 mm tissue culture dishes and incubated at 37°C and 6.5% CO2 to 80–90% confluence. All experiments were performed with HPAEC at 80–90% confluence.
3
Culturing Human Umbilical Vein Endothelial Cells
4
Nrf2 Pathway Activation in HepG2 and HPAEC Cells
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We utilized the human HepG2 cell line (hepatocellular carcinoma) and primary human pulmonary artery endothelial cells (HPAECs) for genomic assays. HepG2 cells and HPAECs are suitable models in the present work because they each have a Nrf2 pathway that responds in a normal manner to Nrf2 activators [33 (link),34 (link)], and do not have reported mutations in Nrf2/Keap1. The HepG2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured and maintained by standard methods, using Opti-MEM medium with 4% fetal bovine serum (FBS) and geneticin/penicillin/streptomycin. HPAECs were procured from Lonza (Morristown, NJ, USA, catalog # CC-2530) and cultured in Endothelial Basal Media-2 (Lonza catalog #: CC-3516) supplemented with endothelial growth factors optimized for aortic and pulmonary arterial endothelial cells (Lonza catalog # CC-3162). HPAEC subculturing was limited to six passages in order to prevent senescence and de-differentiation. HPAECs were seeded at a density of 5 × 105 cells per 100 mm tissue culture dishes and incubated at 37 °C and 6.5% CO2 to 80–90% confluence. All experiments were performed with HPAECs at 80–90% confluence.
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