The human brain endothelial cell line HBEC5i (CDC, Atlanta, USA) was cultured in DMEM/F-12 GlutaMAX medium (Gibco) supplemented with 10 % heat-inactivated fetal bovine serum (Gibco), 1X endothelial cell growth factor (Sigma) and 10U/ml penicillin/streptomycin (Gibco).
Endothelial cell growth factor
Manufactured by Merck Group
Sourced in United States, United Kingdom
Endothelial cell growth factor is a laboratory reagent used to promote the growth and proliferation of endothelial cells in cell culture. It provides the necessary growth factors and nutrients required for the maintenance and expansion of endothelial cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Heparin, by Merck Group (11 mentions)
M199 media, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
M199 medium, by Thermo Fisher Scientific (4 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Merck Group (4 mentions)
Collagenase, by Worthington (3 mentions)
Porcine heparin, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Bovine insulin, by Thermo Fisher Scientific (3 mentions)
Bovine pituitary extract, by Thermo Fisher Scientific (3 mentions)
Hydrocortisone, by Merck Group (3 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Geneticin, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
25 cm2 culture flasks, by Thermo Fisher Scientific (2 mentions)
Dmem f12 glutamax medium, by Thermo Fisher Scientific (1 mentions)
32 protocols using endothelial cell growth factor
1
Culturing Human Brain Endothelial Cells
2
Isolation and Culture of HUVECs
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HUVECs were isolated from umbilical cords from different individuals (n = 178) using a standard procedure (50 (link)). Cords from miscarriages, stillbirths, or HIV positive, group B streptococcus positive or hepatitis B infected mothers were excluded. Cells were maintained in M199 medium (Sigma, M4530) supplemented with 15% foetal bovine serum (Appleton Woods, FB021), l -glutamine, 2.5 μg/ml β-endothelial growth factor (Sigma, E1388), 4.5 μg/ml endothelial cell growth factor (Sigma, E2759), 2.5 μg/ml thymidine (Sigma, 89270), heparin (Sigma, H3393), and 1% penicillin/streptomycin. Cells of passage ≤4 were used for the experiments in this study. HEK293 cells were maintained in DMEM medium (Sigma, D5796) supplemented with 10% foetal bovine serum, 3.34 mM/l l -glutamine, 0.02 units/l penicillin and 20 mg/l streptomycin.
3
Radiation-induced oxidative stress in HUVECs
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Human umbilical vein endothelial cells (HUVEC) were harvested and isolated by enzymatic digestion in the presence of type II collagenase (0.1%) as described before [36 (link)]. Isolated cells were maintained in Medium 199 (Life Sciences, Grand Island, NY, USA), containing fetal bovine serum (10%), antibiotics, and growth factors (heparin, 50 U/mL, and endothelial cell growth factor, 10 mg/mL) (all from Sigma-Aldrich, St. Louis, MO, USA). Human cells were obtained from discarded umbilical cords and treated anonymously; as such, approval from the University Ethics Review Board was not necessary. HUVEC were used at passage 2 after primary culture. Cell monolayers were pretreated with RiduROS (0.1, 1, and 10 μg/mL) or a single compound for 24 h before exposure to a single final dose of 0.25 Gy X-ray radiations with a dose corresponding to maximum oxidative stress generation compared with the control, as previously demonstrated [15 (link)]. Incubation with only DMSO (0.0075%, the final percent to reach the concentration of RiduROS 1 μg/mL) was run in parallel, and no inhibition of oxidative stress was observed with DMSO after irradiation. Control cells were treated exactly as irradiated samples, except for irradiation and/or drug treatments.
4
Culturing HUVEC under Hyperglycemic Conditions
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HUVEC were purchased from the American Type Culture Collection (ATCC Manassas, Virginia, USA) and cultured in medium M199 supplemented with 10% fetal bovine serum, 1 mM l -Glutamine, 1 mM Penicillin–Streptomycin (Euroclone, Milano, Italy), 1 mM Sodium Pyruvate, 5 U/mL Heparin and 150 µg/mL Endothelial Cell Growth Factor on collagen-coated dishes (50 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA). d -glucose was used at concentrations of 11.1 mM and 30 mM and l -glucose (Sigma-Aldrich) was utilized as control of osmolarity (30 mM). 30 mM glucose corresponds to severe hyperglycaemia in diabetic individuals and is used in many studies on EC, whereas 11.1 mM glucose is a pathological concentration rarely used in in vitro experiments.
5
Culture of Human Pancreatic β-cells and HUVECs
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Human pancreatic β-cells (1.1B4) were purchased from Sigma-Aldrich (UK) and cultured in RPMI 1640 medium containing 2 mM L-Glutamine and 10% v/v foetal bovine serum (FBS, Sigma-Aldrich UK). Whereas human umbilical endothelial cells (HUVECs) were grown in a complete cell medium previously prepared by adding 0.05 mg/mL endothelial cell growth factor (Sigma, UK), 0.1 mg/mL sodium heparin (Sigma, UK) and 10% v/v foetal bovine serum (FBS) to the F12K growth medium (Lonza, UK). Both cells were cultured under standard culture conditions (37 °C, 5%CO2) and used at passage lower than 25 (1.1B4 β-cells) and 32 (HUVECs), respectively.
6
Stable Expression of Ackr3b and Ackr3
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COS cells, grown in Dulbecco modified Eagle's medium (DMEM) (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% FBS (Gibco Life Technologies), were transfected using Lipofectamin reagent (Invitrogen, Carlsbad, CA, USA) with a pcDNA vector harboring the ackr3b cDNA under the control of the CMV promoter and selected with geneticin (500 μg/ml; Invitrogen) to obtain ackr3b-COS cells. Stable expression of ackr3b was confirmed by RT-PCR. Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and used at early (I–IV) passages. HUVECs were grown on culture plates coated with porcine gelatin in M199 medium (Gibco Life Technologies), supplemented with 20% FBS, endothelial cell growth factor (10 μg/ml), and porcine heparin (100 μg/ml) (Sigma-Aldrich). Chinese hamster ovary (CHO) cells were transfected with a bicistronic pIRES-EGFP vector harboring the human ACKR3 cDNA (kindly provided by Prof. Marcus Thelen, Institute for the Research Biomedicine, Bellinzona, Switzerland) using Lipofectamin reagent and selected with 350 μg/ml geneticin (Gibco Life Technologies) to obtain ACKR3-CHO cells. Stable expression of ACKR3 was confirmed by RT-PCR and fluorescent analysis of EGFP+ cells (see Figures 9A,B ).
7
Isolation and Culture of Primary HUVECs
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Primary HUVECs were isolated as previously described [8 (link),30 (link)]. In brief, the umbilical cord vein from normal term placentas was cannulated and infused with phosphate buffered saline (PBS; 137 mM NaCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, pH 7.4) to wash out foetal blood. Approximately 10 mL (1 mg/mL) of pre-warmed collagenase (Worthington, Lakewood, NJ, USA) was infused into the cord, followed by incubation at 37 °C for 8 min. HUVECs were dissociated with an infusion of PBS and collected in newborn calf serum (Sigma Aldrich, St. Louis, MO, USA). The dissociated HUVECs were recovered by pelleting and resuspension, followed by culture in M199 media (Life Technologies, CA, USA) containing 20% newborn calf serum, 1% endothelial cell growth factor, 1% heparin (Sigma), and 1% AA, and used between passages 1–3. Cells were then seeded for treatment with M199 media containing 10% fetal calf serum, 1% endothelial cell growth factor, 1% heparin, and 1% AA and incubated under 8% O2, 5% CO2 at 37 °C overnight.
8
Cell Culture Protocols for KSHV-Infection
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L1T2 cells, obtained from ATCC, were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and passaged every 3–4 days. At the onset of this project, L1T2 cells were available for purchase from ATCC but are no longer available despite having been demonstrated to maintain long-term KSHV infection and to induce tumors upon xenotransplantation into immune deficient mice [20 (link)]. TIVE cells, kindly provided by Rolf Renne, were cultured in Medium 199 supplemented with 20% FBS, 1% penicillin/streptomycin, 1% 200 mM L-Glutamine, and 30 mg Endothelial Cell Growth Factor (Sigma, Burlington, MA, USA) and reportedly lack the capacity to produce tumors in immunodeficient mice [19 (link)]. The media were changed two times per week, and the cells were split 1:2 when the culture reached ~60% confluency.
9
Adenovirus-mediated HO-1 Overexpression in HUVEC
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The recombinant adenovirus expressing Hmox1 (Ad-HO-1) and the Ad0 control adenovirus were amplified in human embryonic kidney 293A cells, purified and titrated as described32 (link). HUVEC were infected by incubation with adenovirus in serum-free M199 for 2 h at 37 °C and maintained in M199 containing 10% fetal FBS and 7.5 μg/ml endothelial cell growth factor (Sigma-Aldrich). Optimal multiplicity of infection (MOI) for Ad-HO-1 was previously determined by immunoblotting32 (link).
10
Isolation and Melatonin Treatment of HUVECs
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The cord vein of umbilical cords of normal term placentas was cannulated and infused with PBS to wash out fetal blood. Next, approximately 10ml (1mg/ml) of collagenase (Worthington, Lakewood, New Jersey) was infused into the cord followed by incubation at 37°C for 10 minutes. The dissociated HUVEC cells were recovered by pelleting and resuspension followed by culture in M199 media (Life Technologies) containing 20% fetal calf serum, 1% anti-anti and 1% endothelial cell growth factor (Sigma) and 1% heparin. HUVECs were cultured at 37°C under 20% O2. Isolated primary HUVECs (between passage 2 and 4) were treated with increasing doses of melatonin (Sigma (M5250); 1–1000μM) for 48 h.
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