Human umbilical vein endothelial cells (HUVECs) were acquired from Lonza (cat# CC-2519) and cultured in EBM Basal Medium (Lonza, cat# CC-3121) supplemented with the EGM Singlequots Supplement Pack (Lonza, cat# CC-4133) following the supplier’s recommendations. After thawing quickly in a 30°C water bath, ~600 thousand cells were seeded on a 10 cm tissue culture dish. Cells were split at a 1:2 ratio every 2 days by trypsinization (Lonza cat# CC-5012), followed by centrifugation at 200 × g for 3 min before resuspending and reseeding in EBM Basal Medium supplemented with the EGM Singlequots Supplement Pack. Cells were cultured for not more than 8 passages.
Ebm basal medium
Manufactured by Lonza
Sourced in Switzerland
EBM basal medium is a cell culture medium designed to support the growth and maintenance of endothelial cells. It provides the necessary nutrients and components for the cultivation of these specialized cell types.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Bovine gelatin, by Merck Group (2 mentions)
Human aortic endothelial cells, by Lonza (2 mentions)
Trypsin, by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Growth factor reduced matrigel, by Corning (1 mentions)
Cell culture insert, by BD (1 mentions)
Smgm 2 bulletkit, by Lonza (1 mentions)
Aosmc, by Lonza (1 mentions)
Egm singlequots kit, by Lonza (1 mentions)
Egm 2 singlequot kit supplements and growth factors, by Lonza (1 mentions)
Huvecs, by Lonza (1 mentions)
Elisa brdu colorimetric immunoassay, by Roche (1 mentions)
Cc 2519, by Lonza (1 mentions)
Nitric acid, by Thermo Fisher Scientific (1 mentions)
Sodium citrate tribasic dehydrate, by Merck Group (1 mentions)
Egm endothelial cell growth medium singlequots supplements, by Lonza (1 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (1 mentions)
Geltrex ldev free reduced growth factor basement membrane matrix, by Thermo Fisher Scientific (1 mentions)
Cobalt chloride hexahydrate, by Thermo Fisher Scientific (1 mentions)
11 protocols using ebm basal medium
1
Cultivation of Human Umbilical Vein Endothelial Cells
2
Choroid Explant Angiogenesis Assay
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Adult C57Bl/6 mice were euthanized, and eyes were immediately enucleated and kept in ice-cold EBM basal medium (Lonza) before dissection. Choroid explants were placed in growth factor-reduced Matrigel (Corning) seeded in 24 well plates, and incubated at 37° C for 10 minutes to allow the Matrigel to solidify. 500 μL of medium was then added to each well and incubated at 37° C with 5% CO2 for 24 hours before lentiviral infections. Explant pictures were taken after 48 hours (at the beginning of choroid vessel growth), and at 72 hours to 96 hours post-infection to follow vessel growth. Phase contrast photos of individual explants were captured with a ZEISS Axio Oberver.Z1 microscope. Sprouting area quantification was performed using the semi-automated macro plug-in to the Image J software designed for this purpose [51 (link)].
3
Invasion Assay for Endothelial Cells
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The invasion assays were performed in 24-well plates equipped with cell culture inserts with a transparent PET membrane containing 8 μm pores (BD). One day before the assay, HUVECs were serum-starved in EBM basal medium (Lonza) supplemented with 0.5% FBS. The cells were harvested and resuspended at a concentration of 5 × 105/mL in serum-free medium. Two hundred microliters (1×105 cells) of resuspended cell solution with SMYGT or vehicle (PBS) were added to the inserts pre-coated with diluted Matrigel. EGM-2 complete medium was added to the bottom chamber as a chemo-attractant. Sulforaphane (5 μM) was administered in parallel as a positive control drug. After 16 h, the media were carefully aspirated from the inserts and the membrane filters were fixed with 70% ethanol for 10 min. The cells on the upper surface of the filters were completely removed using cotton swabs, and the invading cells on the opposite surface of the filter were stained with methylene blue. The GAPDH-visualized invading cells in three randomly selected fields per well (× 100 magnification) were digitally captured and counted using an inverted microscope.
4
HiChIP Experiments with HAEC and AoSMC
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HAEC (catalog no. CC-2535) and human AoSMC (catalog no. CC-2571) were obtained from Lonza. The HAEC were grown in endothelial growth medium (EGM medium), which is formulated by mixing the contents of the EGM SingleQuots kit (Lonza catalog no. CC-4133 containing bovine brain extract [BBE], ascorbic acid, hydrocortisone, epidermal growth factor [hEGF], fetal bovine serum [FBS], and gentamicin/amphotericin-B [GA] with EBM basal medium (Lonza catalog no. CC-3121). AoSMC were grown in SmGM-2 BulletKit (Lonza catalog no. CC-3182). The following growth supplements are added to a 500-mL bottle of smooth muscle cell basal medium: hEGF, 0.5 mL; insulin, 0.5 mL; hFGF-B, 1 mL; FBS, 25 mL; GA-1000, 0.5 mL. Both cell types were cultured at 37 °C in 5% CO2. For cell culture expansion, trypsin/EDTA (ethylenediaminetetraacetic acid) was used for detachment of cells. Cells between passage 5 and 7 were used for the following HiChIP experiments.
5
Evaluating Liposome Cytotoxicity on Cancer and Normal Cells
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Murine 4T1 breast cancer cells and human FaDu squamous cell carcinoma cells were obtained from the ATCC. Cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, penicillin (100 IU/mL), streptomycin (100 µg/mL), and amphotericin B (0.25 µg/mL). Normal human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (Basel, Switzerland) and cultured in EBM basal medium supplemented with EGM-2 SingleQuot kit supplements and growth factors (Lonza).
FaDu squamous cell carcinoma cells or 4T1 metastatic breast cancer cells were seeded at a density of 3,000 cells per well in a 96-well plate. HUVECs, which served as control (normal cells), were seeded at a density of 4,000 cells per well in a 96-well plate. After 24 hours, cells were treated with ω-liposomes and C-liposomes at the indicated concentrations for 24 hours. To determine the number of dividing cells, bromodeoxyuridine (BrdU) reagent was added to the cells for 4–6 hours and an ELISA BrdU colorimetric immunoassay (Hoffman-La Roche Ltd, Basel, Switzerland) was performed, according to the manufacturer’s protocol.
FaDu squamous cell carcinoma cells or 4T1 metastatic breast cancer cells were seeded at a density of 3,000 cells per well in a 96-well plate. HUVECs, which served as control (normal cells), were seeded at a density of 4,000 cells per well in a 96-well plate. After 24 hours, cells were treated with ω-liposomes and C-liposomes at the indicated concentrations for 24 hours. To determine the number of dividing cells, bromodeoxyuridine (BrdU) reagent was added to the cells for 4–6 hours and an ELISA BrdU colorimetric immunoassay (Hoffman-La Roche Ltd, Basel, Switzerland) was performed, according to the manufacturer’s protocol.
6
Culturing Primary and Immortalized Endothelial Cells
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Primary human umbilical vein endothelial cells (HUVEC, [35 (link)]) from pooled donors (Lonza, CC-2519) were cultured in gelatine-coated cell culture flasks in EBM basal medium (Lonza) supplemented with EGM singleQuot supplement and growth factors (Lonza). Cells were maintained at 37 °C under 5% CO2 and 95% humidity and experiments were performed with cells in passage 4–9. The hybrid EA.hy926 cell line, generated by fusion of HUVEC with cells of the lung carcinoma cell line A549 [36 (link)], was cultured in 1% gelatine-coated cell culture flasks in DMEM 1965 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were maintained like HUVEC and not used above passage 20.
7
Isolation and Culture of Human Aortic Endothelial Cells
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Human aortic endothelial cells were obtained from Lonza (Walkersville, MD). Upon receipt, the cells were seeded in 75-cm2 flasks precoated with 1% (w/v) bovine gelatin (Sigma-Aldrich) at a ratio of 1:2 and were grown in EBM basal medium (Lonza) containing bovine brain extract, ascorbic acid, hydrocortisone, epidermal growth factor, 2% (v/v) fetal bovine serum (FBS; Sigma-Aldrich), and gentamicin/amphotericin-B at 37 °C under 5% CO2 in a humidified atmosphere. Medium was replaced periodically until cells reached 80–90% confluence; cells were then detached with 0.05% (w/v) trypsin-0.02% (w/v) EDTA (Sigma-Aldrich) and sub-cultured in gelatin-precoated 75 cm2 flasks at a 1:3 ratio. Cells at passage 7 were used for experiments.
8
HUVEC cell lentiviral transduction
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HUVEC cells (LONZA #CC-2519) were grown in EBM™ Basal medium (LONZA, #CC-3121) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). HUVEC cells were co-infected with various lentiviral mixtures, transfected with both plasmids with different gRNAs sites for 72 h. EGFP and mCherry double-positive cells were obtained by flow sorting and were cultured and expanded for 5 generations. After expansion, the HUVEC cells were used in downstream functional analysis.
9
Synthesis of Gold Nanoparticles
10
Isolation and Culture of Human Aortic Endothelial Cells
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Human aortic endothelial cells were obtained from Lonza (Walkersville, MD). Upon receipt, the cells were seeded in 75-cm2 flasks precoated with 1% (w/v) bovine gelatin (Sigma-Aldrich) at a ratio of 1:2 and were grown in EBM basal medium (Lonza) containing bovine brain extract, ascorbic acid, hydrocortisone, epidermal growth factor, 2% (v/v) fetal bovine serum (FBS; Sigma-Aldrich), and gentamicin/amphotericin-B at 37 °C under 5% CO2 in a humidified atmosphere. Medium was replaced periodically until cells reached 80–90% confluence; cells were then detached with 0.05% (w/v) trypsin-0.02% (w/v) EDTA (Sigma-Aldrich) and sub-cultured in gelatin-precoated 75 cm2 flasks at a 1:3 ratio. Cells at passage 7 were used for experiments.
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