Mouse embryonic ECs were isolated as previously described (Fraccaroli et al., 2015 (link)) and cultured in EC growth medium (Promocell). Human umbilical vein endothelial cells (HUVECs) (Pelobiotech) were cultured in EC medium (Promocell). Human dermal microvascular endothelial cells (HMECs) (American Type Culture Collection) were cultured in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 10% EC growth medium (Promocell), 10% fetal calf serum (FCS) and 1% Penicillin/Streptomycin.
Ec growth medium
Manufactured by PromoCell
EC growth medium is a cell culture medium designed to support the growth and maintenance of endothelial cells. It provides the necessary nutrients and growth factors to enable endothelial cell proliferation in vitro.
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Lab products found in correlation
Ec medium, by PromoCell (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
C 12203, by PromoCell (1 mentions)
Dispase 2, by Boehringer Ingelheim (1 mentions)
Collagenase a, by Merck Group (1 mentions)
Human epidermal growth factor (hegf), by Merck Group (1 mentions)
Hdmec, by PromoCell (1 mentions)
Ec growth medium mv, by PromoCell (1 mentions)
Hpaec, by PromoCell (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Hcaec, by PromoCell (1 mentions)
Huvecs, by PELOBiotech (1 mentions)
Hmecs, by American Type Culture Collection (1 mentions)
Fetal calf serum (fcs), by PromoCell (1 mentions)
Sigenome smartpool, by Horizon Discovery (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Human umbilical vein endothelial cells (huvec), by PromoCell (1 mentions)
Gelatin solution, by Merck Group (1 mentions)
25 cm2 culture flasks, by BD (1 mentions)
7 protocols using ec growth medium
1
Isolation and Culture of Endothelial Cells
2
Isolation and Culture of HUVECs
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Human umbilical vein endothelial cells (HUVECs) were purchased from Promocell (C-12203) or alternatively isolated from embryos as described elsewhere24 (link) and cultured with EC medium (Promocell). For the isolation of ECs, embryos were harvested at E13.5 and washed in PBS. For each embryo, the tail was removed and used for genotyping. Embryos were treated with digestion buffer (collagenase A [Sigma; 1 mg/mL] and dispase-II [Boehringer; 1 mg/mL] in PBS) at 37°C for 60 minutes. Samples were filtered through cell strainers and incubated with VE-cadherin–coated Dynabeads for 30 minutes at room temperature. VE-cadherin–positive cells coupled to Dynabeads were purified with a magnet, centrifuged, and resuspended in fresh EC growth medium (Promocell) for culturing.
3
Isolation and Culture of Endothelial Cells
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Ethical approval for endothelial cell isolation and subsequent experimentation was granted by Regionala etikprövningsnämnden i Stockholm (diarienummer 2015/1294-31/2). Human Umbilical Vein Endothelial Cells (HUVEC) were isolated as previously described97 (link), from anonymised umbilical cords collected from Karolinska University Hospital. HUVEC were grown in Medium 199 (M199, Gibco) supplemented with 20% foetal bovine serum (FBS), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 1 μg/ml Hydrocortisone, 1 ng/ml Human Epidermal Growth Factor (all Sigma), and 1.25 μg/ml Amphotericin B (Invitrogen). Human Pulmonary Artery Endothelial Cells (HPAEC), Human Coronary Artery Endothelial Cells (HCAEC), and Human Dermal Microvascular Endothelial Cells (HDMEC) were obtained from Promocell in cryogenically frozen vials, and were cultured in EC Growth Medium (HPAEC, HCAEC) or EC Growth Medium-MV (HDMEC) (Promocell). For some experiments, serum starvation was carried out using 2% or 0.5% FBS.
4
Silencing β1 Integrin and CCM2 in Endothelial Cells
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EA.hy926 cells, obtained from the ATCC (Manassas, USA) were cultured in Dulbecco modified Eagle's medium (DMEM, Invitrogen, France), supplemented with 10% fetal bovine serum, Hepes buffer (10 mM, PAA), glutamine (2 mM, PAA) and antibiotics. Passages 18–26 of EA.hy926 cell line were used. HUVEC cells were purchased from PromoCell, plated on fibronectin coated flasks and cultured in EC growth medium (PromoCell) supplemented with antibiotics. Only early passages of HUVEC (between 2 and 4) were used. For β1 integrin silencing, HUVEC (1.5×106 cells) were transfected three times at 24 h interval with the β1 siRNA (smart pool siGenome, Dharmacon) or CT siRNA (AGG-UAG-UGU-AAU-CGC-CUU-G) at concentration 20 nM by using 45 µl Lipofectamine RNAi max (Invitrogen) according to the manufacturer's instructions. Cells were used the day after the third round of transfection. Silencing efficiency of β1 siRNA was determined by western blot (Fig. 2E ) and by real-time PCR (99±0.5%, N = 3, not shown). For CCM2 silencing, EA.hy926 cells were transfected three times at 24 h interval and HUVEC cells were transfected two times as described previously (Faurobert et al., 2013 (link)). Silencing efficiency of CCM2 siRNA was determined by western blot (Fig. 3D , Fig. 5B ) and by real-time PCR (99±0.4% in EA.hy926 cell line and 95±0.8% in HUVEC, N = 3, not shown).
5
Isolation and Cultivation of Human Umbilical Vein Endothelial Cells
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Human umbilical cords were obtained from the Human Biomaterials Resource Centre (University of Birmingham; ethics 09/H1010/75), who collected fully consented tissue from the Birmingham Women’s Hospital National Health Service Trust. Human umbilical vein EC (HUVEC) were isolated from umbilical cords as previously described (21 (link)) and cultured in M199 supplemented with 20% FCS, 10 ng/ml epidermal growth factor, 35 μg/ml gentamicin, 1 μg/ml hydrocortisone (all from Sigma-Aldrich), and 2.5 μg/ml amphotericin B (Life Technologies Invitrogen Compounds). HUVEC were grown to confluence in 25-cm2 culture flasks (BD Falcon, Oxford, U.K.) precoated with 1% gelatin solution (Sigma-Aldrich). For the monocyte static adhesion and transmigration time-course experiments, primary blood microvascular EC were purchased from PromoCell, cultured in the manufacturer’s recommended medium (EC growth medium; PromoCell), and passaged up to four times prior to use in experiments (20 (link)).
6
HUVEC Transfection and TNF-α Stimulation
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Human umbilical vein ECs (HUVECs) were purchased from PromoCell, plated on cell culture dishes coated with collagen (Biochrom AG), cultured in EC growth medium (PromoCell) according to manufacturer's recommendations and used between passages 5 and 8. Transfection of HUVECs with predesigned ON-TARGETplus SMARTpool human HDAC9 siRNA or nontargeting control (Dharmacon) was conducted by electroporation using the HUVEC Nucleofector Kit (Lonza) following the manufacturer's instructions. Where indicated, plasmid DNA was cotransfected. Cells recuperated for 48 or 72 hours before entering the experiment. HUVECs were stimulated with 20 ng/ mL human TNF-α (tumor necrosis factor-alpha; PeproTech) at different time intervals.
7
Isolation and Cytokine Treatment of CD34+ Cells
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Isolation of mononuclear cells from heparinized blood was performed by Ficoll-Seperating Solution (Biochrom). After isolation, mononuclear cells were washed twice in phosphate-buffered saline (PBS) and re-suspended in PBS. CD34 + ECs were isolated using Flow-Comp-Flexi Dynabeads (Invitrogen) after incubating with anti-CD34-Mouse-Antibody (Invitrogen). Purity of CD34 isolation reached 97%±2,5% as demonstrated by immunofluorescence staining (FITC-labeled anti-CD34, negative control FITC Mouse IgG 1 , κ Isotype Control, both BD Biosciences), of these 90% displayed expression of CD31 (PE-labeled anti-CD31, negative control PE Mouse IgG 1 , κ Isotype Control, both BD Biosciences; data not shown).
Isolated CD34 + cells were either left untreated or incubated with 10ng/ml tumor necrosis factor (TNF)-α or interferon (IFN)-γ in EC growth medium (PromoCell) at 37°C for four hours. Both cytokines have been demonstrated to induce expression of costimulatory and adhesion molecules by CD34 + -cells [26] [27] [28] [29] . Cell viability in cytokine treated cells was analyzed using trypan blue (Thermo Scientific, Pierce Biotechnology, Illinois, USA) following the manufacturer's directions. Viability was >95% in all conditions studied (data not shown).
Isolated CD34 + cells were either left untreated or incubated with 10ng/ml tumor necrosis factor (TNF)-α or interferon (IFN)-γ in EC growth medium (PromoCell) at 37°C for four hours. Both cytokines have been demonstrated to induce expression of costimulatory and adhesion molecules by CD34 + -cells [26] [27] [28] [29] . Cell viability in cytokine treated cells was analyzed using trypan blue (Thermo Scientific, Pierce Biotechnology, Illinois, USA) following the manufacturer's directions. Viability was >95% in all conditions studied (data not shown).
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