Human umbilical vein endothelial cells (HUVECs) were maintained in endothelial basal medium-2 (EBM-2) containing endothelial growth medium-2 (EGM-2 SingleQuots Kit, Lonza). The medium was changed every 3 days. HUVECs were used between passages 1 and 5.
Egm 2 singlequots kit
Manufactured by Lonza
Sourced in Switzerland, United States, Germany
The EGM-2 SingleQuots Kit is a laboratory product that provides a single-use, pre-measured cocktail of essential growth factors and supplements for the culture of endothelial cells. The kit is designed to facilitate the preparation of complete growth medium for endothelial cell lines.
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Lab products found in correlation
Huvecs, by Lonza (7 mentions)
Ebm 2, by Lonza (3 mentions)
Ebm 2 basal medium, by Lonza (2 mentions)
Mes sa, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (2 mentions)
Hcaec, by Lonza (2 mentions)
β estradiol, by Merck Group (2 mentions)
Matrigel matrix, by BD (1 mentions)
Pe conjugated annexin 5, by BD (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Lonza (1 mentions)
L glutamine, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
Endothelial cell basal medium 2 ebm 2, by Lonza (1 mentions)
R3 igf 1, by Lonza (1 mentions)
Huvecs, by RIKEN BioResource Center (1 mentions)
Fgm 2 bulletkit, by Lonza (1 mentions)
Hbmec, by ScienCell (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
22 protocols using egm 2 singlequots kit
1
Culturing Human Umbilical Vein Endothelial Cells
2
Platelet activation and angiogenesis
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Acid-soluble thrombin receptor PAR1 activating peptide (PAR1-AP) was from Calbiochem (Darmstadt, Germany). Water-soluble PAR1-AP and PAR4 activating peptide (PAR4-AP), prostaglandin I2 (PGI2), and cultured cell detach solution (0.01% trypsin/5 mM EDTA) were purchased from Sigma (St Louis, MO, USA). Platelets were identified by the fluorescein isothiocyanate (FITC)-conjugated CD42a MAb Beb 1 (Becton Dickinson; San Jose, CA, USA). Platelet P-selectin expression was determined by R-phycoerythrin (PE)-CD62P MAb, and platelet phosphotidylserine (PS) exposure was monitored by PE-conjugated annexin V (both from Becton Dickinson). Immunoassay kits monitoring platelet release of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF, BB type), platelet factor 4 (PF4), and thrombospondin-1 (TSP-1) were from R&D Systems Ltd (Abingdon, UK). Endothelial culture media (EBM-2 Basal Medium and the EGM-2 SingleQuots kit) and fetal bovine serum (FBS) were purchased from Lonza (Basel, Switzerland). Matrigel ™ Matrix was from Becton Dickinson.
3
Glioblastoma Cell Lines and HUVECs Culture
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U87MG-luc2 GBM cell line was purchased from Caliper Life Science (A PerkinElmer Company, Hopkinton, MA, USA) and cultured in Eagle's Minimum Essential Medium (EMEM) (Gibco, Invitrogen, Carlsbad, CA, USA). U251, U373 and U343 GBM cell lines were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD, USA) and cultured in high-glucose (4500 mg l−1) Dulbecco's Modified Eagle's Medium (DMEM) (Lonza, Basel, Switzerland). EMEM and DMEM were supplemented with 10% heat-inactivated foetal bovine serum (FBS), 1% L -glutamine (2 mM) and 1% penicillin/streptomycin (50 units ml−1), all from Sigma-Aldrich. Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and maintained in Endothelial Cell Basal Medium-2 (EBM-2, Lonza). Each 500 ml of EBM-2 was supplemented with EGM-2 SingleQuots Kit (Lonza) consisting the following growth supplements: human epidermal growth factor (hEGF), 0.5 ml; vascular endothelial growth factor (VEGF), 0.5 ml; R3-insulin-like growth factor-1 (R3-IGF-1), 0.5 ml; ascorbic acid, 0.5 ml; hydrocortisone, 0.2 ml; human fibroblast growth factor-beta (hFGF-β), 2.0 ml; heparin (0.5 ml); fetal bovine serum (FBS), 25.0 ml; gentamicin/amphotericin-B (GA), 0.5 ml. All cells were maintained at 37 °C in humidified air with 5% CO2.
4
Hyperglycemia Effects on HUVEC Cells
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Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Lonza,
Breda, The Netherlands) and maintained in EGM-2 media consisting of endothelial
basal medium-2 supplemented with growth factors and antibiotics (EGM-2
SingleQuots kit, Lonza, The Netherlands). The effect of hyperglycemia on HUVECs
was investigated by incubating cells in EGM-2 media with 30 mM D-glucose. The
normal EGM-2 media used as a control contained 5.5 mM D-glucose. In all
experiments, cells from passage 4 or 5 were used and cultured at 37°C in a 5%
CO2/95% humidified atmosphere.
Breda, The Netherlands) and maintained in EGM-2 media consisting of endothelial
basal medium-2 supplemented with growth factors and antibiotics (EGM-2
SingleQuots kit, Lonza, The Netherlands). The effect of hyperglycemia on HUVECs
was investigated by incubating cells in EGM-2 media with 30 mM D-glucose. The
normal EGM-2 media used as a control contained 5.5 mM D-glucose. In all
experiments, cells from passage 4 or 5 were used and cultured at 37°C in a 5%
CO2/95% humidified atmosphere.
5
Establishing Sarcoma and Endothelial Cell Lines
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Human uterine sarcoma cell line MES-SA was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MES-SA/Dx5 is a multidrug-resistant subline derived from MES-SA. Human umbilical vein endothelial cells (HUVECs) were procured from the Bioresource Collection and Research Center, Food Industry Research and Development Institute (BCRC, Hsinchu, Taiwan). MES-SA/Dx5 cells were maintained in McCoy’s 5A medium supplemented with 10% FBS, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin and 0.4 μg/mL of DOX. HUVECs were grown in M199 medium containing endothelial growth medium (EGM)-2 SingleQuots Kit (consisting of FBS, VEGF and other growth factors) (Lonza, Basel, Switzerland), 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL penicillin and 100 μg/mL streptomycin. All the cells were cultured at 37 °C in a humidified incubator with 95% air and 5% CO2.
6
Culturing Human Vascular and Lung Cells
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Human umbilical vein endothelial cells (HUVECs, Lonza) were cultured in endothelial basal medium-2 supplemented with EGM-2 SingleQuots Kit (EGM-2, Lonza) and passages 3 to 5 were used for the experiments. Normal human lung fibroblasts (LFs, Lonza) were cultured in fibroblast basal medium supplemented with FGM-2 BulletKit (FGM-2, Lonza) and passages 5 to 7 were used for the experiments. In terms of HUVECs, cells were cultured for experiments before reaching 80 to 90% of confluent. All cells were incubated in a humidified 5% CO2 atmosphere and at 37 °C.
7
Culturing HUVEC and MES-SA Cells
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Human umbilical vein endothelial cells (HUVECs, BCRC H-UV001) were obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). Human uterine sarcoma cell line MES-SA (CRL-1976) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The multidrug-resistant human uterine sarcoma cell line MES-SA/Dx5 was established from MES-SA cells in the presence of increasing DOX concentrations, as described previously [45 (link)]. HUVECs were cultured in M199 medium containing the endothelial growth medium (EGM)-2 SingleQuots Kit (consisting of FBS, VEGF and other growth factors) (Lonza, Basel, Switzerland), 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin. MES-SA/Dx5 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.4 μg/mL of DOX. All cell lines were maintained at 37 °C in a humidified incubator at 5% CO2.
8
Human Brain Microvascular Endothelial Cell Culture
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Human BMECs (HBMECs) were purchased from ScienCell (Cat. No. 1000) and cultured in endothelial cell growth medium 2 (EGM-2; Lonza, CC-3156) supplemented with the EGM-2 SingleQuots™ kit (Lonza, CC-4176), 20% fetal bovine serum, and 1% penicillin/streptomycin (Cat. No. 0503). Cells were routinely passaged at 80–90% confluency, and cells between passages 3 and 6 were used for experiments. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
9
In Vitro Culturing of Cell Lines
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HCT116 and CCD-18Co cells were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea). Human umbilical vein endothelial cells (HUVECs) were obtained from the Lonza (San Diego, CA, USA). HCT116 cells were cultured in McCoy’s medium (Gibco Cell Culture, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Gibco). CCD-18Co cells were cultured in MEM (Gibco) with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco), and were used between passages 5 and 6. HUVECs were grown in EBM-2 (Lonza) supplemented with an EGM™-2 SingleQuots™ kit (Lonza), and used between passages 2 and 4 for experiments. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. A hypoxia incubator (New Brunswick Scientific, Edison, NJ, USA) containing 1% O2, 5% CO2, and 94% N2 was used to create hypoxic conditions.
10
Cultivation of Primary Human Umbilical Vein Endothelial Cells
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Commercially available primary human umbilical vein endothelial cells (HUVEC, Lonza, Cologne, Germany) were cultivated under static cell culture conditions (37 °C, 5 vol.-% CO2) in tissue culture polystyrene flasks (TCP, Techno Plastic Products AG, Trasadingen, Switzerland) with endothelial basal medium EBM-2 supplemented with EGM-2 Single Quots® kit and 2 vol.-% FBS to EGM-2 full medium (Lonza, Cologne, Germany). HUVEC were used for no longer than four passages [49 (link)]. All HUVEC and all culture media and culture media supplements were from the same charges. HUVEC identity was proved by immune fluorescent detection of von Willebrand factor expression (Figure 8 ).
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