Egm 2 medium

Manufactured by Lonza

Sourced in Switzerland, United States, United Kingdom, France, Belgium, Japan, Germany

EGM-2 medium is a cell culture medium that supports the growth and maintenance of endothelial cells. It contains the necessary supplements and growth factors to promote the proliferation and survival of endothelial cells in vitro.

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Close spelling variants of this product

EGM-2 (912 citations) Endothelial Growth Medium-2 (EGM-2; (37 citations) EGM-2 culture medium (12 citations) EGMTM-2 Endothelial Cell Growth Medium-2 BulletKitTM (11 citations) EGM-2-MV culture medium (7 citations) EGMTM-2 Bullet Kit medium (6 citations) EGM-2 cell culture medium (6 citations) EGMTM -2 MV Microvascular Endothelial Cell Growth Medium (4 citations) EGMTM-2 MV Microvascular Endothelial Cell Growth Medium-2 BulletKitTM (4 citations) EGMTM-2 MV Medium (3 citations)

322 protocols using egm 2 medium

Alveolar Epithelial-Endothelial Bilayer Model

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A model of the alveolar epithelium/endothelium was constructed on transwell membranes as described by Hope et al.[12] (link). Briefly, Human pulmonary artery endothelial cells (HPAEC) and Human alveolar epithelial cells (A549) were obtained from Lonza. The bilayer was constructed by incubating 1×10⁵ HPAECs on the under surface of a 6.5 mm diameter Transwell Clear membrane with 3-µM pores (Corning) for 2 h. The membrane was then placed right side up into the well of a 24-well tissue culture plate (Greiner) containing EGM-2 medium (Lonza), the upper chamber was filled with 100 ul EGM-2 medium and the assembly was incubated for 24 h at 37°C and 5% CO₂. The contents of the upper chamber were removed and replaced by 5×10⁴ A549 cells in 100 µl EBM-2 plus 10% FBS, the assembly was then incubated at 37°C and 5% CO₂. The respective media were changed daily and the experiment performed on the fifth day following addition of the A549 cells.

Morton C.O., Fliesser M., Dittrich M., Mueller T., Bauer R., Kneitz S., Hope W., Rogers T.R., Einsele H, & Loeffler J. (2014). Gene Expression Profiles of Human Dendritic Cells Interacting with Aspergillus fumigatus in a Bilayer Model of the Alveolar Epithelium/Endothelium Interface. PLoS ONE, 9(5), e98279.

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Spheroid Invasion Assay for Angiogenesis

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Three × 10³ cells were incubated in 150 µl medium containing 20% methylcellulose (M‐0512, Sigma‐Aldrich) in round bottom microtitre plates to allow spheroid formation within 48 hours. Spheroids were washed in EGM2 medium (Lonza, Walkersville, MD), transferred to confluent BEC or LEC monolayers expressing mCherry and kept in EGM2 medium; 3 hours (Hep3B), 4 hours (HCC‐1.2) or 5.5 hours (HepG2) later, the spheroid and the gap area in the BEC/LEC monolayer underneath were photographed. For each condition, the 2D size of > 10 spheroids and appendant gaps were determined by ImageJ software (National Institutes of Health). Further detail see.¹¹

Paur J., Valler M., Sienel R., Taxauer K., Holzmann K., Marian B., Unterberger A., Mohr T., Berger W., Gvozdenovich A., Schimming J., Grusch M, & Grasl‐Kraupp B. (2020). Interaction of FGF9 with FGFR3‐IIIb/IIIc, a putative driver of growth and aggressive behaviour of hepatocellular carcinoma. Liver International, 40(9), 2279-2290.

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Evaluating pNaKtide effects on retinal cells

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Human retinal microvascular endothelial cells (HRMECs) and human retinal pigment epithelium (ARPE-19) cells were purchased from Cell Systems (CSC 2M1; Kirkland, WA, USA) and American Type Culture Collection (CRL-2302; Manassas, VA, USA), respectively. EGM-2 Medium and HyClone Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12) were purchased from Lonza (CC-3162; Basel, Switzerland) and Cytiva (SH30023.01; Marlborough, MA, USA), respectively. HRMECs were cultured in EGM-2 Medium supplemented with growth factors (CC-4176, Lonza), 5% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. ARPE-19 cells were cultured in DMEM-F12 media supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were kept at 37°C in humidified air with 5% CO₂. HRMECs and ARPE-19 cells were treated with or without pNaKtide or a scrambled peptide with a Tat leader sequence (Tat-scrambled peptide, sequences listed in Supplementary Table S1), at a concentration of 0.5, 1, or 5 µM for 24 hours. A Trypan blue exclusion assay was performed to evaluate cell viability.

Wang J., Wang X., Gao Y., Lin Z., Chen J., Gigantelli J., Shapiro J.I., Xie Z, & Pierre S.V. (2020). Stress Signal Regulation by Na/K-ATPase As a New Approach to Promote Physiological Revascularization in a Mouse Model of Ischemic Retinopathy. Investigative Ophthalmology & Visual Science, 61(14), 9.

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Culturing Human Umbilical Vein ECs and Fibroblasts

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Human umbilical vein ECs were purchased from Lonza and cultured in EGM2 medium (Lonza) with changes of the medium every 2 d. Human fibroblasts were purchased from ScienCell Research Lab and cultured in Dulbecco’s modified Eagle’s medium (Gibco), supplemented with 20% FBS and 1% filter-sterilized penicillin–streptomycin.^{59 (link)}.

Sayed N., Huang Y., Nguyen K., Krejciova-Rajaniemi Z., Grawe A.P., Gao T., Tibshirani R., Hastie T., Alpert A., Cui L., Kuznetsova T., Rosenberg-Hasson Y., Ostan R., Monti D., Lehallier B., Shen-Orr S.S., Maecker H.T., Dekker C.L., Wyss-Coray T., Franceschi C., Jojic V., Haddad F., Montoya J.G., Wu J.C., Davis M.M, & Furman D. (2021). An inflammatory aging clock (iAge) based on deep learning tracks multimorbidity, immunosenescence, frailty and cardiovascular aging. Nature aging, 1, 598-615.

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Expansion of Human Endothelial Cells

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HUVECs were obtained from Lonza (Basel, Switzerland) and expanded in EGM2 medium (Lonza) containing 5% fetal bovine serum, antibiotics, insulin-like growth factor, ascorbic acid, hydrocortisone, and angiogenic factors, including basic fibroblast growth factor, epidermal growth factor, and vascular endothelial growth factor (VEGF).

Yang X., Zhu L., Tada S., Zhou D., Kitajima T., Isoshima T., Yoshida Y., Nakamura M., Yan W, & Ito Y. (2014). Mussel-inspired human gelatin nanocoating for creating biologically adhesive surfaces. International Journal of Nanomedicine, 9, 2753-2765.

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Isolation and Characterization of EPCs

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Hs294T human melanoma cells and prostate cancer cells (PC3) were purchased from ATCC and cultured in DMEM supplemented with 10% FCS at 37°C in 5% CO₂ humidified atmosphere. Endothelial progenitor cells (EPCs) have been isolated from human umbilical cord blood as previously described [44 (link),45 (link)]. EPCs were cultured on gelatin 1% coated dishes in EGM-2 medium (Lonza). Hs294T cells were transfected with RacN17 (a dominant negative mutant of Rac1) or EphA2 constructs using Lipofectamine 2000 (Invitrogen) according to manifacturer’s instructions.

Taddei M.L., Giannoni E., Morandi A., Ippolito L., Ramazzotti M., Callari M., Gandellini P, & Chiarugi P. (2014). Mesenchymal to amoeboid transition is associated with stem-like features of melanoma cells. Cell Communication and Signaling : CCS, 12, 24.

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Immunocytochemistry of APJ Receptor

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HREC cells were seeded on four chamber Lab-Tek Chambered Coverglass (Thermo Scientific; 1500 cells/chamber) for cell imaging in EGM-2 medium (Lonza) in a 5% CO₂ atmosphere at 37°C for 1 day. Chambers were washed with ice-cold PBS and fixed for 10 min with paraformaldehyde (4%) on ice then washed in PBS containing Triton X-100 (0.2% v/v) for 2 x 20 min to permeabilize the cells. To block nonspecific protein-protein interactions, cells were washed with Odyssey Blocking Buffer and PBS (1:1 v/v) containing 5% BSA for 1 h at room temperature then incubated with rabbit anti -APJ antibodies (Abcam; ab66218, ab84296, ab140508; 1:500 dilution, 2μg/ml) overnight at +4°C. Goat anti-rabbit Alexa Fluor 594 secondary IgG (H+L; Invitrogen, 1:500 dilution, 4μg/ml) and DAPI Fluoromount-G (SouthernBiotech) were used for anti-APJ receptor antibodies labeling and cell nuclei staining for confocal fluorescence microscopy detection.

McAnally D., Siddiquee K., Gomaa A., Szabo A., Vasile S., Maloney P.R., Divlianska D.B., Peddibhotla S., Morfa C.J., Hershberger P., Falter R., Williamson R., Terry D.B., Farjo R., Pinkerton A.B., Qi X., Quigley J., Boulton M.E., Grant M.B, & Smith L.H. (2018). Repurposing antimalarial aminoquinolines and related compounds for treatment of retinal neovascularization. PLoS ONE, 13(9), e0202436.

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Differentiation of ieCPCs into CMs, SMCs, and ECs

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For the generation of CMs, ieCPCs were seeded onto Matrigel-coated plates at a density of 3 × 10⁵ cells/cm² and kept in serum-free differentiation (SFD) medium for 10 days. In some experiments, 5 μM IWP2 was included during the first 6 days to increase the yield of CMs. For the generation of SMCs and ECs, ieCPCs were seeded onto Matrigel-coated plates at a density of 1 × 10⁴ cells/cm² and cultured in SFD medium supplemented with 2 ng/ml TGF-β1 and 10 ng/ml PDGF-BB (for SMCs) or in EGM-2 medium (Lonza) (for ECs) for 10 days.

Zhang Y., Cao N., Huang Y., Spencer C.I., Fu J.D., Yu C., Liu K., Nie B., Xu T., Li K., Xu S., Bruneau B.G., Srivastava D, & Ding S. (2016). Expandable Cardiovascular Progenitor Cells Reprogrammed from Fibroblasts. Cell stem cell, 18(3), 368-381.

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Culturing Human Endothelial and Breast Cancer Cells

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Human umbilical vein endothelial cells (HUVECs) were obtained from IRCCS MultiMedica, (Milan, Italy) and were cultured in Endothelial Growth Medium-2 (#CC-3156; EGM™-2 Medium, Lonza, Basel, Switzerland) supplemented with EGM™ SingleQuots™ Kit (#CC-4176 Lonza, Basel, Switzerland). HUVECs were maintained in culture until passage 6. Human TNBC cell lines MDA-MB-231 and BT-549 were purchased from American Type Culture Collection (ATCC, VA, USA) and cultured in DMEM and RPMI-1640 medium respectively (#BE12-604F; #BE12-702F Lonza, Basel, Switzerland) supplemented with 10% FBS (#ECS5000L Euroclone), 100 units/ml of penicillin and 100 mg/ml of streptomycin (#ECB3001_3380; Euroclone). In addition, RPMI-1640 medium was supplemented with 0,023 units/ml of insulin. Cells were routinely tested for the presence of Mycoplasma by Hoechst stain (#62249; ThermoFisher). All cells were kept in a humidified incubator with 5% CO2 at 37°C.

Scagliotti A., Capizzi L., Cazzaniga M.E., Ilari A., De Giorgi M., Cordani N., Gallazzi M., Bruno A., Pelosi G., Albini A., Lavitrano M., Grassilli E, & Cerrito M.G. (2022). Co-targeting triple-negative breast cancer cells and endothelial cells by metronomic chemotherapy inhibits cell regrowth and migration via downregulation of the FAK/VEGFR2/VEGF axis and autophagy/apoptosis activation. Frontiers in Oncology, 12, 998274.

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Hypoxia-induced Angiogenesis Modeling

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HUVECs (Lonza, Walkersville, MD, USA) were cultured with EGM-2 medium (Lonza) and maintained at 37 °C with 5% CO₂. Hypoxia was performed by incubating cells in an incubator with 1% O₂ (Thermo, Middletown, VA, USA). Human PlGF and VEGF-E were purchased from ProSpec (East Brunswick, NJ, USA). Cycloheximide and chloroquine were purchased from Tocris (Bristol, UK), bafilomycin-A (BafA) was from Sigma (St. Louis, MO, USA). HeLa cells were obtained from ATCC (Manassas, VA, USA) and cultured in 10% FBS (ThermoFisher Scientific, Waltham, MA, USA) containing DMEM (ThermoFisher Scientific).

Hong J., Min Y., Wuest T, & Lin P.C. (2020). Vav1 is Essential for HIF-1α Activation via a Lysosomal VEGFR1-Mediated Degradation Mechanism in Endothelial Cells. Cancers, 12(6), 1374.

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