Patients undergoing aortocoronary artery bypass surgery were recruited from the Department of Cardiac Surgery at Leeds Teaching Hospitals. Primary saphenous vein (SV) endothelial cells (SVECs) were isolated from segments of human SV, obtained as previously described (18 (link)). Ethical approval was granted by the local Research Ethics Committee (Ref. No. CA01/040). SVECs were grown in EBM-2 growth medium supplemented with an EGM-2 bullet kit (Lonza) and used up to passage 3.
Ebm 2 growth medium
Manufactured by Lonza
Sourced in United States
The EBM-2 growth medium is a cell culture medium designed to support the growth and maintenance of embryonic stem cells. It provides the necessary nutrients and growth factors to maintain the undifferentiated state of these cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Hmecs, by Lonza (2 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Rat tail collagen 1, by BD (1 mentions)
N ethyl n isopropylpropan 2 amine, by Merck Group (1 mentions)
4 4 dithiodibutyricacid, by Merck Group (1 mentions)
N n dimethylpyridin 4 amine dmap, by Merck Group (1 mentions)
Ac 500 mhz, by Bruker (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Roche (1 mentions)
Huvecs, by American Type Culture Collection (1 mentions)
Egm 2mv bulletkit, by Lonza (1 mentions)
Crl 3243, by American Type Culture Collection (1 mentions)
Human keratinocyte growth supplement, by Thermo Fisher Scientific (1 mentions)
Ebm 2 basal medium, by Lonza (1 mentions)
Epilife medium, by Thermo Fisher Scientific (1 mentions)
Cm0149, by Thermo Fisher Scientific (1 mentions)
Trypsin solution, by Merck Group (1 mentions)
Human ascs, by Lonza (1 mentions)
Singlequot, by Lonza (1 mentions)
M199 medium, by Thermo Fisher Scientific (1 mentions)
8 protocols using ebm 2 growth medium
1
Isolation of Human Saphenous Vein Endothelial Cells
2
Synthesis and Characterization of Small Molecule Inhibitors
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Abacavir,
darunavir, and nelfinavir were
provided by the NIH AIDS Reagent Program (Germantown, MD). ((1H-Benzo[d][1,2,3]triazol-1-yl)oxy)tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (PyBOP) was bought from GenScript Corporation
(Piscataway, NJ). DTT was purchased from Roche (Indianapolis, IN).
1-(3-Dimethylaminoproyl)-3-ethylcarbodiimide hydrochloride (EDC) was
purchased fom AK Scientific (Union City, CA). 4,4′-Dithiodibutyric
acid, N,N-dimethylpyridin-4-amine
(DMAP), and N-ethyl-N-isopropylpropan-2-amine
(DIEA) were purchased from Sigma-Aldrich (St. Louis, MO). hCMEC/D3
cells were donated from Institut National de la Sante et de la Recherche
Medicale (INSERM, Paris, France). Rat tail collagen I was purchased
from BD Biosciences (San Jose, CA). EBM-2 growth medium was purchased
from Lonza (Basel, Switzerland). Calcein-AM was bought from Invitrogen
(Carlsbad, CA), All other reagents were purchased from Sigma-Aldrich
(St. Louis, MO) or Invitrogen (Carlsbad, CA) and used without further
purification. 1H, 13C, and 2D NMR spectra were
recorded with a Bruker AC 800 and Bruker AC 500 MHz. DMSO-d6 was used to prepare NMR samples.
darunavir, and nelfinavir were
provided by the NIH AIDS Reagent Program (Germantown, MD). ((1H-Benzo[d][1,2,3]triazol-1-yl)oxy)tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (PyBOP) was bought from GenScript Corporation
(Piscataway, NJ). DTT was purchased from Roche (Indianapolis, IN).
1-(3-Dimethylaminoproyl)-3-ethylcarbodiimide hydrochloride (EDC) was
purchased fom AK Scientific (Union City, CA). 4,4′-Dithiodibutyric
acid, N,N-dimethylpyridin-4-amine
(DMAP), and N-ethyl-N-isopropylpropan-2-amine
(DIEA) were purchased from Sigma-Aldrich (St. Louis, MO). hCMEC/D3
cells were donated from Institut National de la Sante et de la Recherche
Medicale (INSERM, Paris, France). Rat tail collagen I was purchased
from BD Biosciences (San Jose, CA). EBM-2 growth medium was purchased
from Lonza (Basel, Switzerland). Calcein-AM was bought from Invitrogen
(Carlsbad, CA), All other reagents were purchased from Sigma-Aldrich
(St. Louis, MO) or Invitrogen (Carlsbad, CA) and used without further
purification. 1H, 13C, and 2D NMR spectra were
recorded with a Bruker AC 800 and Bruker AC 500 MHz. DMSO-d6 was used to prepare NMR samples.
3
Culturing Human Umbilical Vein Endothelial Cells
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Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC (Rockville, MD, USA) and cultured in endothelial basal medium-2 (EBM-2) growth medium (Lonza, Walkersville, MD, USA), containing hydrocortisone, epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor-1, VEGF, ascorbic acid, heparin, and 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2. The cells were seeded in culture flasks or on plates coated with 1% gelatin and allowed to grow to confluence before experimental treatment. The HUVECs used in the experiment were between passages 3 and 5. The commercially available vascular endothelial cell-specific supplement EGM®-2 MV Bullet Kit (Lonza) was used (19 (link)).
4
In Vitro Diabetic Retinal Cell Culture
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A human retinal pericyte (HRP) line was stabilized in our laboratory [14] (link). Human microvascular endothelial cells (HMECs) were purchased from ATCC (Cat#CRL-3243, RRID:CVCL_0307), and the human Müller cell line Moorfields/Institute of Ophthalmology-Müller 1 (MIO-M1) (RRID:CVCL_0433) obtained from the UCLB licensing portal XIP (London, UK) [15] . HRP and MIO-M1 cells were cultured in DMEM+10%FCS; HMEC in EBM-2 growth medium (Lonza) for expansion and DMEM+10%FCS for experiments.
Cells were cultured for 8 days in physiological conditions (NG, 5.6 mmol/l D-glucose), or intHG (48hr high glucose, 28 mmol/l /48hr NG, twice), to better mimic the diabetic microenvironment, since we had previously demonstrated that human pericytes are more affected by intermittent high glucose conditions [16] (link). 50 µmol/l thiamine (T) and 100 µmol/l fenofibric acid (FA) (concentrations chosen on the basis of previous reports [7, (link)10] (link)) were added during the whole 8-day incubation. Hypoxic conditions (hypo) were obtained by keeping cultures in a 5%CO 2 / 94%N 2 /1%O 2 gas mixture for the last 48 hrs.
Reagents for cell cultures were purchased from Sigma-Aldrich, unless otherwise stated.
Cells were cultured for 8 days in physiological conditions (NG, 5.6 mmol/l D-glucose), or intHG (48hr high glucose, 28 mmol/l /48hr NG, twice), to better mimic the diabetic microenvironment, since we had previously demonstrated that human pericytes are more affected by intermittent high glucose conditions [16] (link). 50 µmol/l thiamine (T) and 100 µmol/l fenofibric acid (FA) (concentrations chosen on the basis of previous reports [7, (link)10] (link)) were added during the whole 8-day incubation. Hypoxic conditions (hypo) were obtained by keeping cultures in a 5%CO 2 / 94%N 2 /1%O 2 gas mixture for the last 48 hrs.
Reagents for cell cultures were purchased from Sigma-Aldrich, unless otherwise stated.
5
Culturing and Co-culturing Skin Cells
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Primary normal human epidermal keratinocytes were cultured at 37°C in 5% CO2 in Epilife medium supplemented with human keratinocyte growth supplement (Gibco, USA). Human microvascular endothelial cells (HMVECs) were cultured at 37°C in 5% CO2 in EBM-2 basal medium supplemented with EBM-2 growth medium (Lonza, USA).
M. furfur (ATCC 12078) was cultured at 30°C on Difco YM agar supplemented with 1% olive oil. S. epidermidis (Staphylococcus epidermidis, ATCC 12228) was cultured at 37°C on Difco tryptic soy agar. C. acnes (Cutibacterium acnes, ATCC 6919) was cultured at 37°C on forced clostridial medium (CM0149; Oxoid) with 2% agar. To induce hypoxia, a BD GasPakTM EZ Pouch was used. All the media were sterilized by autoclaving at 121°C for 15 min.
Organisms were harvested by centrifugation, and the pellet was suspended in the corresponding media. The organisms were heat-killed by incubation at 80°C for 3 min, and then co-cultured with normal human epidermal keratinocytes or human microvascular endothelial cells for 24 h at a density of 1 × 105 cells/mL. To induce allergic environments, recombinant thymic stromal lymphopoietin (TSLP) (50 ng/mL) or IL-4 (50 ng/mL) was used.
M. furfur (ATCC 12078) was cultured at 30°C on Difco YM agar supplemented with 1% olive oil. S. epidermidis (Staphylococcus epidermidis, ATCC 12228) was cultured at 37°C on Difco tryptic soy agar. C. acnes (Cutibacterium acnes, ATCC 6919) was cultured at 37°C on forced clostridial medium (CM0149; Oxoid) with 2% agar. To induce hypoxia, a BD GasPakTM EZ Pouch was used. All the media were sterilized by autoclaving at 121°C for 15 min.
Organisms were harvested by centrifugation, and the pellet was suspended in the corresponding media. The organisms were heat-killed by incubation at 80°C for 3 min, and then co-cultured with normal human epidermal keratinocytes or human microvascular endothelial cells for 24 h at a density of 1 × 105 cells/mL. To induce allergic environments, recombinant thymic stromal lymphopoietin (TSLP) (50 ng/mL) or IL-4 (50 ng/mL) was used.
6
Isolation and Culture of Human Mesenchymal and Epithelial Cells
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Human ASCs were obtained from Lonza (Basel, Switzerland), cultured in complete MSCGM™ Mesenchymal Stem Cell Growth Medium (Lonza) containing, 1% antibiotic–antimycotic (HyClone) at 37°C in 5% CO2 incubator. When homogeneous monolayer with typical fibroblast morphology was obtained, ASCs were passaged using trypsin solution (Sigma, St. Louis, MO, USA). For the experiments, cells of the 4–6 passages were used. At these passages phenotypic characterization and functional evaluation of mesenchymal properties were performed as previously described [55 (link)].
HMECs were purchased from Lonza (Basel, Switzerland) and were cultured in EBM-2 growth medium (Lonza) supplemented with a cocktail of angiogenic factors (SingleQuots, Lonza) according to the instructions of the manufacturer.
HMECs were purchased from Lonza (Basel, Switzerland) and were cultured in EBM-2 growth medium (Lonza) supplemented with a cocktail of angiogenic factors (SingleQuots, Lonza) according to the instructions of the manufacturer.
7
HUVEC Culture and Shear Stress Analysis
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Human umbilical vein endothelial cells (HUVECs) were purchased from Thermo Fisher Scientific Inc. (Thermo Fisher Scientific, Waltham, MA, USA). HUVECs were cultured in M199 medium (Thermo Fisher Scientific) supplemented with 20% (v/v) fetal bovine serum (FBS), 10% (v/v) EBM-2 growth medium (Lonza, MD, USA), 100 U/mL penicillin/streptomycin, and then incubated in a growth chamber at 37°C with CO2 (5%, v/v).
Glass slides were coated with collagen prior to being seeded with HUVECs (1 × 10 3 cells/cm 2 ). After incubation in a growth chamber for 24 hours, the HUVECs were either harvested (static treatment) or exposed to SF with 12 dynes/cm 2 for 1 hour in an SF device.
For RSV treatments, HUVECs were incubated with 10 μM RSV (Tocris Bioscience, MN, USA) for 1 hour.
Glass slides were coated with collagen prior to being seeded with HUVECs (1 × 10 3 cells/cm 2 ). After incubation in a growth chamber for 24 hours, the HUVECs were either harvested (static treatment) or exposed to SF with 12 dynes/cm 2 for 1 hour in an SF device.
For RSV treatments, HUVECs were incubated with 10 μM RSV (Tocris Bioscience, MN, USA) for 1 hour.
8
Establishing Human Retinal Pericyte and Endothelial Cell Lines
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A stabilized human retinal pericyte line (HRPs) is currently available in our laboratory (Berrone et al., 2009) (link), while human microvascular ECs (HMECs) were purchased from Lonza (Basel, Switzerland). HRPs were cultured in DMEM + 10% FCS, while HMECs in EBM-2 growth medium (Lonza). When used in the experiments, both HRPs and HMECs were maintained in DMEM + 10% FCS. Reagents for cell cultures were purchased from Sigma-Aldrich (St Louis, MO, USA).
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