PRB-Ishikawa cells were generously provided by Dr. Leen Blok (Erasmus University, Netherlands).11 (link) These cells were maintained in DMEM/F12 supplemented with 10% FBS, sodium pyruvate, penicillin/streptomycin, hygromycin, and G418. PRB-Ishikawa cells were sequenced and authenticated by Dr. Christopher Korch at the University of Colorado Cancer Center (UCCC) DNA Sequencing & Analysis Core. Primary uterine microvascular endothelial cells were purchased from Lonza (Basel, Switzerland) and maintained in Endothelial Growth Media supplemented with 2% FBS, hEGF, VEGF, R3-IGF-1, Ascorbic Acid, Hydrocortisone, hFGF-β, Heparin, and GA. MK-2206 was generously provided by Merck Sharp & Dohme Corp (Kenilworth, NJ, USA) and the National Cancer Institute, National Institutes of Health (Bethesda, MD, USA).
Endothelial growth media
Manufactured by Lonza
Sourced in Switzerland, United States
Endothelial growth media is a specialized cell culture medium designed to support the growth and maintenance of endothelial cells in vitro. It provides the necessary nutrients and growth factors to promote the proliferation and survival of endothelial cells, which are an important component of the vascular system.
Automatically generated - may contain errors
Lab products found in correlation
Primary uterine microvascular endothelial cells, by Lonza (2 mentions)
Mk2206, by Merck Group (2 mentions)
M199 media, by Merck Group (2 mentions)
Aria 2 flow cytometer, by BD (2 mentions)
Collagenase 2, by Merck Group (2 mentions)
Dnase, by Worthington (2 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
Cd34 hspc, by Lonza (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Texas red conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Hrp linked anti rabbit and anti mouse igg, by Cell Signaling Technology (1 mentions)
Amifostine, by Merck Group (1 mentions)
Alexa fluor 488 labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ve cadherin antibody, by Santa Cruz Biotechnology (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Lysopc, by Avanti Polar Lipids (1 mentions)
Human pulmonary artery endothelial cells, by Lonza (1 mentions)
Collagenase, by BD (1 mentions)
Collagenase, by Merck Group (1 mentions)
11 protocols using endothelial growth media
1
Characterization of Ishikawa Cell Line
2
Characterization of Ishikawa Cell Line
3
Isolation and Characterization of Human Adipose-Derived Vessel-Forming Progenitors
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Human adipose lipoaspirates were obtained after informed consent from female patients (26–54 years old). Fresh lipoaspirates were separated from blood, and lipoaspiration fluid were passed through a 100 μm nylon mesh. Solid matter was then resuspended in collagenase digest buffer (2.2 mg/mL Collagenase II [Sigma] supplemented with 100 U/mL DNase [Worthington] in M199 Media [Sigma]), shaken in CERTOMAT BS-1 Incubation-Shaking Cabinet at 37 °C for 30 minutes, sedimented on Histopaque-1119 Density gradient to remove blood and dead cells, washed, and pelleted at 400×g at 4 °C. Cells were then plated on Lonza Endothelial Growth Media and transduced with CMV-GFP expressing lentivirus. Twenty-four hours after transduction, cells were lifted with collagenase, resuspended in staining media (2% FBS in PBS), blocked with rat IgG, and stained with fluorochrome-conjugated antibodies against human CD45, Tie2, PDGFRα, and CD31 for FACS on the BD Aria II Flow Cytometer. As with mouse samples, the gating was adjusted based on compensation, fluorescence minus one controls and, unstained cells. In ischemic rescue studies, 500 000 sorted VSPC1+VSPC2 vessel-forming progenitors were injected around ligated femoral artery of 8- to 12-week-old immunodeficient Rag2/gamma(c) knockout male mice.
4
Establishment and Culture of hBMEC and HSPC
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Human BMEC (hBMEC) used in all experiments were established by Dr. Graca Almeida-Porada (Wake Forest School of Medicine; Winston-Salem, NC) [66 (link)]. CD34+ HSPC were purchased from Lonza, Inc. (Walkersville, MD). hBMEC were grown on 0.2% gelatin-coated (Biocoat) flasks (Thermo Fisher Scientific Inc. Rockford, IL, USA) in endothelial growth media (Lonza Inc.Walkersville, MD USA) supplemented with 10% fetal bovine serum (FBS). hBMEC were used at ≥75% confluency. HSPC were maintained in HPGM (Lonza, Inc.) supplemented with IL-3, stem cell factor, thrombopoietin, and Flt-3 ligand(HSPC media) (Peprotech, Inc. Rocky Hill, NJ, USA).
5
Isolation and Culture of Rat Nerve Cells
6
Endothelial Cell Responses to Particulate Matter
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Human pulmonary artery endothelial cells and endothelial growth media were obtained from Lonza (Allendale, NJ). Cells were used at passages 5–8 and all cell stimulations were carried out in medium containing 2% fetal bovine serum unless otherwise specified. Texas Red-conjugated phalloidin and Alexa Fluor 488-labeled secondary antibodies were purchased form Molecular Probes (Eugene, OR). Antibodies to phospho-VE-cadherin (pTyr-658 and pTyr-731) were obtained from Invitrogen (Carlsbad, CA) and VE-cadherin antibody was from Santa Cruz Biotechnology (San Jose, CA). p120-Catenin antibody was from BD biosciences (San Diego, CA) and HRP-linked anti-mouse and anti-rabbit IgG were obtained from Cell Signaling (Beverly, MA). N-acetyl cysteine and amifostine were obtained from Sigma (St. Louis, MO). For PM, we used an urban PM 1649b collected from ambient air in Washington, DC and characterized by National Institute of Standards and Technology (certification date: 12/17/2015; expiration date: 07/31/2030). Purified POVPC, PGPC and lyso-PC were obtained from Avanti Polar Lipids (Alabaster, AL).
7
Isolation and Characterization of Vessel-Forming Progenitors
8
Silencing VEGFR2 and VEGFR3 in HUVECs
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HUVECs were transferred into endothelial growth media (Lonza) for 24 h, then cells at ∼75% confluency were transfected with 10 nM final ON-Target Smartpool siRNAs (L-003138 and L-003148 from GE Healthcare; AM4636 from Ambion) complexed with RNAiMAX (Invitrogen). VEGFR2 siRNA, 5′-GGGCAUGUACUGACGAUUA-3′, 5′-CUACAUUGUUCUUCCGAUA-3′, 5′-GGAAAUCUCUUGCAAGCUA-3′, and 5′-GCGAUGGCCUCUUCUGUAA-3′; VEGFR3 siRNA, 5′-CGCCCGAGUUCCAGUGGUA-3′, 5′-GAACUUGACCGACCUCCUG-3′, 5′-GCGAAUACCUGUCCUACGA-3′, and 5′-GCAAGAACGAUCUGUU-3′. Cells were maintained in transfection media for 24 h, then returned to standard M199 base media. Experiments were performed 72–90 h after transfection. For adenoviral rescue, cells were infected with virus and 5 µg/ml polybrene 36 h after siRNA transfection.
9
Culturing Human Umbilical Vein Endothelial Cells
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Primary human umbilical vein endothelial cells (HUVECs, pooled vein donor samples) cultured in endothelial growth media plus (Lonza Ltd.) at 37°C at 5% C02. HUVEC–derived EA.hy926 cells were cultured using 10% FBS and Dulbecco’s Modified Eagles Media at 37°C at 5% C02.
10
Culturing Human Umbilical Vein Endothelial Cells
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