BECs grown in EGM2 complete medium for 1 day were used for this experiment. After washing with HBSS, cells were treated either with vehicle (DMSO) or 17β-Oestradiol (10μM) for 16 hrs in Glucose-, Glutamine- and serum-free endothelial cell medium (cell biologics) and supplemented either with 5mM Glucose, 2mM Glutamine or 20mM Linoleic acid-Oleic acid (Sigma) to determine the effect of nutrient supplementation on cell growth. Cells were stained with anti-Ki-67 (Abcam, 1:300) to measure EC proliferation or with cleaved caspase-3 (Cell Signalling Technology, 1:300) to measure apoptosis.
Endothelial cell medium (ecm)
Manufactured by Cell Biologics
Sourced in United States
Endothelial cell medium is a specialized cell culture medium designed to support the growth and maintenance of endothelial cells. It provides the necessary nutrients and growth factors required for the in vitro culture of endothelial cells derived from various sources, such as human umbilical vein, aorta, or microvascular tissues.
Automatically generated - may contain errors
Lab products found in correlation
Linoleic acid oleic acid, by Merck Group (2 mentions)
Anti ki67, by Abcam (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Endothelial cell growth factors, by Cell Biologics (2 mentions)
Smgm 2 medium, by Lonza (2 mentions)
Hpasmc, by Lonza (2 mentions)
Gelatin based coating solution, by Cell Biologics (1 mentions)
Fetal bovine serum (fbs), by Cell Biologics (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Ms column, by Miltenyi Biotec (1 mentions)
Cd31 microbead, by Miltenyi Biotec (1 mentions)
Ra 6023, by Cell Biologics (1 mentions)
Tumor necrosis factor (tnf), by Thermo Fisher Scientific (1 mentions)
Tumor necrosis factor (tnf), by Merck Group (1 mentions)
Pf 562 271, by Merck Group (1 mentions)
11 protocols using endothelial cell medium (ecm)
1
Nutrient Effects on Endothelial Cell Growth
2
Culturing Mouse Fibroblasts and Endothelial Cells
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Two kinds of cells were used for in vitro experiments. Mouse embryonic fibroblasts (MEFs) were prepared from E17.5 Zeb1+/+, Zeb1+/−, and Zeb1−/− embryos40 (link), whereas mouse retinal microvascular endothelial cells (mRMVECs) were purchased from Cell Biologics (Cat. #: C57–6065). They were cultured in DMEM medium with 10% fetal bovine serum (FBS) and Endothelial Cell Medium (Cell Biologics cat. #: M1168), respectively, under 5% CO2 at 37 °C. The above media were mixed with 1% penicillin and streptomycin antibiotics and refreshed every 3 days until cell confluence.
3
Isolation and Culture of Primary Vascular Cells
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Mouse and rat PVEC (mPVEC and rPVEC) were purchased from Cell Biologics and maintained in Endothelial Cell Medium containing 5% fetal bovine serum (FBS), endothelial cell growth factors, and antibiotics (Cell Biologics). Mouse and rat PASMC (mPASMC and rPASMC) were isolated from mouse lungs as we described previously26 and were maintained in SmGM‐2 medium (Lonza) containing 5% FBS, growth factors, and 1% penicillin‐streptomycin. We also isolated mPASMC from mice exposed to 10% O2 for 3 weeks and from mice exposed to room air for 3 weeks (controls).
Human pulmonary artery endothelial and smooth muscle cells (hPVEC and hPASMC) were purchased from Lonza and were maintained in EBM‐2 and SmGM‐2 medium containing FBS and growth factors (Lonza), respectively.26 All cells were maintained in a humidified incubator with a constant supply of 5% CO2 at 37°C.
Human pulmonary artery endothelial and smooth muscle cells (hPVEC and hPASMC) were purchased from Lonza and were maintained in EBM‐2 and SmGM‐2 medium containing FBS and growth factors (Lonza), respectively.
4
Mouse Brain Microvascular Endothelial Cell Culture
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Mouse primary brain microvascular endothelial cells (MBEC) were purchased from Cell Biologics, IL, United States (Catalog# C57-6023; Lot# 070613T2MP) and cultured according to the manual, but with no heparin (as it interferes with EV uptake Atai et al., 2013 (link); Christianson et al., 2013 (link)). Briefly, T25 flasks were pre-coated with Gelatin-based coating solution (Cell Biologics), 106 cells seeded in the Endothelial Cell Medium (Cell Biologics) and passaged 1:2 upon confluence. Low passages (1–4) have been used in this study.
5
Isolation and Cultivation of Pulmonary Cells
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Mouse pulmonary artery smooth muscle cells (mPASMCs) were isolated from mouse lungs, as we described previously,12 (link) and were maintained in SmGM-2 medium (Lonza, Walkersville, MD) containing 5% fetal bovine serum (FBS), growth factors, and 1% penicillin-streptomycin. We also isolated mPASMCs from mice exposed to 10% O2 for 3 weeks and from mice exposed to room air for 3 weeks (controls). The purity of isolated PASMCs was confirmed by immunostaining of α-SMA (Figure S7 ).
Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from Lonza. hPASMCs isolated from normal human lungs (donor lungs that could not be used) and from lungs of PH patients were provided by the Pulmonary Hypertension Breakthrough Initiative (PHBI). Patient or donor clinical information was described previously.12 (link) These cells were maintained in SmGM-2 medium containing FBS and growth factors (Lonza).12 (link) Use of these cells was approved by the University of Illinois at Chicago Institutional Review Board.
Mouse and rat pulmonary vascular endothelial cells (mPVECs and rPVECs) were purchased from Cell Biologics (Chicago, IL) and maintained in Endothelial Cell Medium containing 5% FBS, endothelial cell growth factors, and antibiotics (Cell Biologics).
All cells were maintained in a humidified incubator with a constant supply of 5% CO2 at 37°C.
Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from Lonza. hPASMCs isolated from normal human lungs (donor lungs that could not be used) and from lungs of PH patients were provided by the Pulmonary Hypertension Breakthrough Initiative (PHBI). Patient or donor clinical information was described previously.12 (link) These cells were maintained in SmGM-2 medium containing FBS and growth factors (Lonza).12 (link) Use of these cells was approved by the University of Illinois at Chicago Institutional Review Board.
Mouse and rat pulmonary vascular endothelial cells (mPVECs and rPVECs) were purchased from Cell Biologics (Chicago, IL) and maintained in Endothelial Cell Medium containing 5% FBS, endothelial cell growth factors, and antibiotics (Cell Biologics).
All cells were maintained in a humidified incubator with a constant supply of 5% CO2 at 37°C.
6
Hypoxia-Induced CXCL1 Expression in Murine Pulmonary Endothelial Cells
7
Rab7a Knockdown in Primary mBECs
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Primary mBECs were purchased from Cell Biologics and grown in endothelial cell medium supplemented with growth factors and 5% FBS (Cell Biologics). Stealth RNAi duplexes targeting Rab7a were purchased from Invitrogen. Stealth RNAi negative control Med CG Duplex #3 (Invitrogen) was used as negative control in all experiments. 12.5 nM of siRNA was transfected using Lipofectamine 2000 (Invitrogen), following the manufacturer’s instructions.
8
Endothelial Cell Stimulation and Analysis
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Mouse aortic endothelial cells (MoAoECs) were cultured in Endothelial Cell medium (CellBiologics Inc., Chicago, IL, USA). Pooled human umbilical vein endothelial cells (HUVECs) were cultured in Endothelial Cell medium-2 (EGM-2 BulletKit) (Lonza, Basel, Switzerland). Cells were maintained at 37 °C in a 5% CO2, 95% air humidified atmosphere and sub-cultured at a dilution of 1:3. All experiments were performed between passage 3 and passage 8.
Stimulation and starvation of endothelial cells were carried out in starvation medium for 2 h; consisting of basal growth medium with supplements and 0.5% fetal bovine serum (FBS). Cells were stimulated in starvation medium with 30 ng mL−1 of mouse recombinant tumor necrosis factor α (TNF-α) together with 40 ng mL−1 of mouse recombinant interleukin 1β (IL-1β) (R&D Systems, Bio-techne, Minneapolis, MN, USA), or 10 ng mL−1 human TNF-α alone for 6 or 24 h. Non-stimulated cells in starvation medium were used as control. The same cytokine stimulation protocol was used to study protein expression analysis by Western blot, flow cytometry and confocal imaging.
Stimulation and starvation of endothelial cells were carried out in starvation medium for 2 h; consisting of basal growth medium with supplements and 0.5% fetal bovine serum (FBS). Cells were stimulated in starvation medium with 30 ng mL−1 of mouse recombinant tumor necrosis factor α (TNF-α) together with 40 ng mL−1 of mouse recombinant interleukin 1β (IL-1β) (R&D Systems, Bio-techne, Minneapolis, MN, USA), or 10 ng mL−1 human TNF-α alone for 6 or 24 h. Non-stimulated cells in starvation medium were used as control. The same cytokine stimulation protocol was used to study protein expression analysis by Western blot, flow cytometry and confocal imaging.
9
Isolation of Cardiac Microvascular Endothelial Cells
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Animals were treated according to approved protocols and animal welfare regulations of the Institutional Animal Care and Use Committee of the Medical College of Georgia, Augusta University. Cardiac microvascular endothelial cells (CMECs) were isolated from the hearts of male C57BL/6 (WT) and diabetic (db/db) mice (The Jackson Laboratory, Bar Harbor, ME, USA) using the enzyme dissociation method. Briefly, mice were euthanized, and ventricular cardiac tissues were minced into 1 mm3 size and digested with 0.1% collagenase IV and 1 U/mL dispase in DMEM medium. Then, the cells were subjected to CD31 positive magnetic-activated cell sorting (MACS) using CD31 microBeads in combination with MS columns (Miltenyi Biotec Inc, Auburn, CA). The isolated CD31-positive cells were plated on fibronectin/gelatin coated wells (0.5 mg fibronectin in 100 ml 0.1% gelatin) and cultured in endothelial cell medium (Cell Biologics, Chicago, IL) containing 5% fetal bovine serum, VEGF, heparin, EGF, ECGS, hydrocortisone, L-glutamine and antibiotic-antimycotic.
10
Investigating FAK Inhibitor Effects on RBMEC
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Primary RBMECs (CellBiologics; Cat No. RA-6023) were cultured in endothelial cell medium (CellBiologics) supplemented with 2% fetal bovine serum, endothelial cell growth factors, and antibiotics (CellBiologics; Cat No. M1266-Kit). Media was changed every 2 days until cells reached confluence. All experiments were conducted between passages 4–6. In each experiment, cultures exposed to TNF for 6 h (10 ng/ml; Thermo Fisher Scientific) were compared with PBS control conditions. The FAK inhibitor, PF-562,271 (FAK-I; 0.5–2.5 μM Sigma Aldrich, St. Louis, MO) was added to RBMECs for 1 h before TNF addition for 6 h.
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