Human coronary artery EC (HCAEC, Cambrex, Verviers, Belgium) proliferation was determined using a BrdU incorporation assay kit (cell proliferation ELISA, Roche, Mannheim, Germany). Briefly, HCAEC were seeded on 1 % gelatin-coated on 96-well plates at a seeding density of 3 × 103 cells per well in endothelial growth medium (EGM-2, Cambrex) for 24 h, followed by serum starvation and pre-treatment with different concentrations of thalidomide between 50 and 250 μg/ml for 24 h. Afterwards cells were stimulated with EGM-2 for 18 h again in the presence or absence of thalidomide in same concentrations. BrdU labeling solution was applied for another 6 h and BrdU incorporation was detected according to the manufacturer’s instructions. Number of viable cells in growth factor enriched buffer alone was set to a value of 100 %. Number of viable cells resulting from different thalidomide concentrations was compared to the number of cells in DMSO-enriched buffer.
Hcaec
Manufactured by Cambrex
Sourced in United States
HCAEC is a type of lab equipment used for cell culture applications. It is designed to maintain and support the growth of human coronary artery endothelial cells (HCAEC) in a controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
Egm 2m medium, by Cambrex (2 mentions)
Tissue culture flasks, by BD (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Tnf α, by Merck Group (1 mentions)
Cc 3156, by Cambrex (1 mentions)
Egm 2mv single quots, by Cambrex (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Bullet kit, by Cambrex (1 mentions)
H9c2 cell, by American Type Culture Collection (1 mentions)
Hpaec, by Cambrex (1 mentions)
Recombinant human il 1β, by Thermo Fisher Scientific (1 mentions)
Hcaec, by Lonza (1 mentions)
Egm 2, by Cambrex (1 mentions)
Cell proliferation elisa, by Roche (1 mentions)
Microplate reader, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
10 protocols using hcaec
1
Thalidomide Inhibits HCAEC Proliferation
2
Activation of Molt-3 T cells and HCAEC
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Molt-3 T cells were obtained from the American Type Culture Collection (Rockville, MD). They were maintained using RPMI1640 media containing 100 μg/l of penicillin/streptomycin along with 10% heat-inactivated fetal bovine serum (FBS). Tissue culture flasks (75-cm2; Falcon, Fisher, Pittsburgh, PA) were used to culture T cells at 37°C under a saturating humidified atmosphere of 95% air and 5% CO2. Phorbol 12-myristate-13-acetate or PMA (0.2 μM; Sigma, St. Louis, MO) was used to activate these cells. HCAEC (Clonetics, San Diego, CA) were propagated and maintained using medium provided by the vendor in Tissue culture flasks (75-cm2; Falcon, Fisher, Pittsburgh, PA). HCAEC were activated with 20 ng/ml of TNF-α (Millipore Sigma) for 24 h.
3
Differentiation of HL-60 Cells into Neutrophils
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HL‐60 cells (BCRC No. 60,027) were maintained in Iscove's modified Dulbecco's medium supplemented with 20% fetal bovine serum, 4 mM L‐glutamine, and 1.5 g/L sodium bicarbonate and incubated at 37°C in 5% CO2. Then, as suggested in a previous study (Walsh, 2009 ), HL‐60 cells were differentiated into neutrophil‐like cells by stimulation with 1.3% DMSO (Sigma‐Aldrich). Human coronary endothelial cells (HCAEC, CC‐2585, Clonetics, Lonza) were maintained in EBM‐2 medium (CC‐3156, Clonetics, Lonza) supplemented with EGM‐2 MV SingleQuots (CC‐4147, Clonetics, Lonza) and incubated at 37°C in 5% CO2.
4
Aging Effects on Transgenic Murine Models
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Wild-type C57BL6 strain (WT) were purchased from Charles River Laboratory. Transgenic mice with overexpression of human Trx (Trx-Tg) or dominant-negative Trx (dnTrx-Tg) were bred and maintained in the animal facility of Texas Tech University Health Sciences Center and have been described previously [25 (link)]. Both males and females were used in this study. All mice strains used in this study are from a C57BL/6 background and are 20-26 months of age, equivalent to human age of 70-75 years [44 (link)]. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Texas Tech University Health Sciences Center and were consistent with the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health. HCAEC were purchased from Clonetics and propagated in endothelial basal medium supplemented with additives (Bullet kit, Clonetics). H9c2 cells were purchased from ATCC and propagated in DMEM with 10% FBS.
5
Endothelial Cell Response to RSPO3 and IL-1β
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Human coronary artery endothelial cells (HCAEC), human pulmonary artery endothelial cells (HPAEC), human cardiac microvascular endothelial cells (HCMVEC), human dermal microvascular endothelial cells (HDMVEC, all from Clonetics, Lonza), and human brain microvascular endothelial cells (HBMVEC, Cell Systems) were propagated as described (Skaria et al., 2017a (link)) and treated with recombinant human RSPO3 (doses ranging from 250 to 1000 ng/mL, PeproTech) and recombinant human IL-1β (20 U/mL, PeproTech) as given in the supplementary methods (Additional file 1 : Supplementary methods ). For details on endothelial cell characterization, see supplementary methods.
6
Culturing Human Coronary Artery Endothelial Cells
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Human coronary artery endothelial cells (HCAEC) were purchased from Cambrex BioScience (Walkersville, Maryland, USA). Cells were plated into 6-well plates (TPP, Trasadigen, Switzerland) at 37°C in a humidified atmosphere at 5% CO2 and grown in EGM-2M medium containing 5% fetal bovine serum, following the manufacturer's instructions (Cambrex BioScience, Walkersville, MD, USA).
7
HCAEC Culture and Stimulation
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HCAEC from four different donors were purchased from Lonza (Walkesville, Maryland, USA). The cells were plated onto 25 cm2 flasks and 6 or 24 well-plates (TPP, Trasadigen, Switzerland) and cultured at 37°C in a humidified atmosphere at 5% CO2. HCAEC were grown in EGM-2M medium (CambrexBioScience, Walkesville, Maryland, USA) containing 5% fetal bovine serum. For experiments, subconfluent cell cultures were used between passages 4 and 6 in serum-free medium, with pretreatment of OLE for 45 min, followed by addition of SAA for 24 h, unless otherwise indicated. Prior to experiments, cells were incubated in serum-free medium for 2 h.
8
Oxidized LDL in Human Coronary Artery ECs
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Human coronary artery ECs (HCAECs, Clonetics, USA) were maintained from passages 4 to 7 in microvascular endothelial cell growth medium (EGM-MV) supplemented with 20% fetal bovine serum and antibiotics (100 μg/ml streptomycin, 100 IU/ml penicillin, and 0.25 μg/ml amphotericin B). Oxidized LDL was prepared as previously described [11 (link),12 (link)], and precautions were taken to prevent endotoxin contamination. The protein concentration of each LDL preparation was determined by using the Lowry method, and thiobarbituric acid–reactive substances (TBARS) were determined as a measure of oxidative lipid modification [11 (link),12 (link)].
9
Assess Homocysteine-Induced Endothelial Cell Viability
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Human coronary artery endothelial cells (ECs; HCAECs, Clonetics, US), at passages 4 to 7, were maintained in EGM-MV medium supplemented with 20% fetal bovine serum (FBS) and antibiotics. For the experiments, all cultures of subconfluent HCAECs were incubated with phosphate-buffered saline (PBS, as a control), homocysteine, cysteine, or electronegative L5 low-density lipoprotein (LDL) isolated from STEMI patients according to a previously described protocol [25 (link), 26 (link)].
Cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
The chemical MTT was purchased from Sigma (St. Louis, MO) [26 (link), 27 (link)]. HCAECs (5 × 104 cells/well) were dispensed into 24-well plates and incubated for 24 h after the addition of homocysteine or different treatments, and the index of EC viability was determined by the colorimetric MTT (tetrazolium) assay. The absorbance was measured at a wavelength of 540 nm for viable cells using a microplate reader (Thermo Electron, Waltham, MA).
Cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
The chemical MTT was purchased from Sigma (St. Louis, MO) [26 (link), 27 (link)]. HCAECs (5 × 104 cells/well) were dispensed into 24-well plates and incubated for 24 h after the addition of homocysteine or different treatments, and the index of EC viability was determined by the colorimetric MTT (tetrazolium) assay. The absorbance was measured at a wavelength of 540 nm for viable cells using a microplate reader (Thermo Electron, Waltham, MA).
10
Culturing HCAECs with Zinc Supplementation
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HCAECs (Human coronary artery endothelial cells) were purchased from Clonetics Corporation (Lonza) and cultured in endothelial basal medium EBM, supplemented with EGM-2/MV SingleQuots with or without addition of 50 µM ZnSO 4 . This concentration has been already defined by others as moderately excessive and does not have a significant effect on the growth or morphology of endothelial cells (Bobilya et al., 2008) (link).
HCAECs were plated at a seeding density of 2,500 cells/cm 2 in T 25 flasks and passaged serially. Harvested cells were counted using the Trypan blue viability stain. Population doublings (PD) were calculated using the formula (log 10 UCY -log 10 I) x 3.32 (where UCY is the number of cells at the end of the passage and I is the number of cells initially seeded). Cells were collected for immediate assessment by flow cytometry or for RNA extraction for subsequent 4 analysis of gene expression. Total RNA was extracted from HCAECs using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. RNA concentration and purity were measured using a NanoDrop spectrometer and samples were stored at -80 °C for measurement using RT-PCR.
HCAECs were plated at a seeding density of 2,500 cells/cm 2 in T 25 flasks and passaged serially. Harvested cells were counted using the Trypan blue viability stain. Population doublings (PD) were calculated using the formula (log 10 UCY -log 10 I) x 3.32 (where UCY is the number of cells at the end of the passage and I is the number of cells initially seeded). Cells were collected for immediate assessment by flow cytometry or for RNA extraction for subsequent 4 analysis of gene expression. Total RNA was extracted from HCAECs using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. RNA concentration and purity were measured using a NanoDrop spectrometer and samples were stored at -80 °C for measurement using RT-PCR.
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