Egm media

Manufactured by Lonza

Sourced in United States

EGM media is a specialized cell culture medium designed for the growth and expansion of endothelial cells. It provides the essential nutrients and growth factors required for endothelial cell proliferation and maintenance in in vitro cell culture experiments.

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Lab products found in correlation

Glutamine, by Merck Group (2 mentions) Penicillin streptomycin, by Merck Group (2 mentions) Pcdna3.1 v5 his topo expression vector, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Low serum growth kit, by American Type Culture Collection (1 mentions) Primary human dermal fibroblasts, by American Type Culture Collection (1 mentions) L929 mouse fibroblasts, by American Type Culture Collection (1 mentions) Egm 2, by Lonza (1 mentions) Rat anti mouse cd31 antibody clone mec13, by BD (1 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions) Dynabeads, by BD (1 mentions) Dmem media, by American Type Culture Collection (1 mentions) Fetal bovine serum (fbs), by Avantor (1 mentions) Cc 2935, by Lonza (1 mentions) Collagen coated flasks, by Corning (1 mentions) Penicillin streptomycin, by Corning (1 mentions) Nod scid, by Jackson ImmunoResearch (1 mentions) Anti 4e bp1, by Cell Signaling Technology (1 mentions)

12 protocols using egm media

Cell Culture Protocols for Fibroblasts and Endothelial Cells

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Cells were maintained at 37 °C with 5% CO₂. Primary human dermal fibroblasts (ATCC, Manassas, VA, USA) were cultured in fibroblast basal medium supplemented with a low serum growth kit containing 2% serum (ATCC). L929 mouse fibroblasts (ATCC) were maintained in Minimum Essential Media supplemented with 10% horse serum and 1% penicillin-streptomycin. Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC) were maintained in EGM-2 and EGM media (2% serum; Lonza, Walkersville, MD), respectively.

McConnell K.I., Shamsudeen S., Meraz I.M., Mahadevan T.S., Ziemys A., Rees P., Summers H.D, & Serda R.E. (2016). Reduced Cationic Nanoparticle Cytotoxicity Based on Serum Masking of Surface Potential. Journal of biomedical nanotechnology, 12(1), 154-164.

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Establishing In Vitro BBB Model

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In this study, human umbilical endothelial cells (HUVEC) were also used, in order to obtain a coculture together with astrocytes as an experimental in vitro model of BBB. HUVEC were purchased from ATCC®. Cells were cultured in EGM Media (Lonza) supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Sigma-Aldrich), and 2 mM glutamine (Sigma-Aldrich) at 37°C in a humidified atmosphere of 95% air, 5% CO₂. For the experiments, 1 × 10⁵ HUVEC cells/cm² were plated in the apical compartment of 6.5 mm Transwells with a 0.4 μm pore size polyester membrane (Corning Costar, Sigma).

Molinari C., Morsanuto V., Ghirlanda S., Ruga S., Notte F., Gaetano L, & Uberti F. (2019). Role of Combined Lipoic Acid and Vitamin D3 on Astrocytes as a Way to Prevent Brain Ageing by Induced Oxidative Stress and Iron Accumulation. Oxidative Medicine and Cellular Longevity, 2019, 2843121.

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Porcine Endothelial Cell and PBMC Isolation

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Porcine aortic endothelial cells (PAECs) were isolated from GTKO A- and O-type blood group pig aortas as previously described (18 (link)) and cultured in EGM media (Lonza Inc., Allendale, NJ, USA). Porcine peripheral blood mononuclear cells (PBMCs) were isolated from GTKO pig blood by density gradient centrifugation using Ficoll–Hypaque. Human embryonic kidney 293 cells (HEK) and HEK cells expressing the porcine B4GALNT2 cDNA were grown in DMEM media with 10% FBS at 37°C in a 5% CO₂ incubator. The full length pig B4GALNT2 open reading frame was amplified from the original library isolated clone using a primers set containing a Kozak consensus sequence and in-frame translation stop signals respectively (Forward: 5′- ACCATGACTTCGTACAGCCCTAG-3′, Reverse: 5′- CAGATACCTTAGGTGGCACATTGGAG-3′). The PCR product was inserted into pcDNA3.1/V5-His TOPO expression vector (Life Technologies Paisley, UK) and transfected into HEK cells using Lipofectamine-2000 (Life Technologies Paisley, UK). A stable G418 resistant HEK clone expressing the porcine B4GALNT2 genes (HEK-B4T) was established and used for further real-time RT-PCR, immunohistochemical staining, complement dependent cytotoxicity, and flow cytometry analysis.

Byrne G.W., Du Z., Stalboerger P., Kogelberg H, & McGregor C.G. (2014). Cloning and expression of porcine β1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen. Xenotransplantation, 21(6), 543-554.

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Endothelial Cell Culture Conditions

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BAECs were grown in DMEM containing 10% FBS and penicillin/streptomycin. HUVECs were grown in DMEM/F12, 10% FBS, 5 mg ml⁻¹ ECGS, 100 μg ml⁻¹ heparin, penicillin/streptomycin or EGM media (Lonza). Mouse endothelial cells were grown in DMEM containing 20% FBS, 1× non-essential amino acids (Gibco), 2 mM L-glutamate, 50 μg ml⁻¹ gentamicin, 4 μg ml⁻¹ amphotericin B, 100 μg ml⁻¹ heparin, 5 mg ml⁻¹ ECGS and penicillin/streptomycin. Primary mouse endothelial cells were isolated from the lung, using rat anti-mouse CD31 antibody (clone MEC13.3, Pharmingen, no. 553370) and Dynabeads (cat. no. 110.35, Invitrogen) as previously described^{64 (link)}. No cell lines were used in this study.

Yun S., Budatha M., Dahlman J.E., Coon B.G., Cameron R.T., Langer R., Anderson D.G., Baillie G, & Schwartz M.A. (2016). Interaction between integrin α5 and PDE4D regulates endothelial inflammatory signalling. Nature cell biology, 18(10), 1043-1053.

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Cell Culture of MCF7, HeLa, and HUVEC

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MCF7 cells (HTB-22, ATCC) and HeLa cells (CCL-2, ATCC) were cultured in DMEM media (ATCC) supplemented with 10% fetal bovine serum (FBS, VWR) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Human umbilical vein endothelial cells (HUVEC, CC-2935, Lonza) were cultured in EGM⁺ media (Lonza) supplemented with 1% penicillin/streptomycin using collagen-coated flasks (Corning). All cells were housed in 5% CO₂ humidified atmosphere at 37 °C. Cell lines were authenticated with STR profiling and tested negative for mycoplasma contamination prior to the study.

Nguyen H.V., Jiang Y., Mohapatra S., Wang W., Barnes J.C., Oldenhuis N.J., Chen K.K., Axelrod S., Huang Z., Chen Q., Golder M.R., Young K., Suvlu D., Shen Y., Willard A.P., Hore M.J., Gómez-Bombarelli R, & Johnson J.A. (2021). Bottlebrush Polymers with Discrete Sidechains Display Stereochemistry- and Conformation-Dependent Biological Properties. Nature chemistry, 14(1), 85-93.

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Establishing HMLER Breast Cancer Model

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HMLER cell line was kindly provided by Robert Weinberg (Whitehead Institute for Biomedical Research of Massachusetts Institute of Technology). HMLER(CD44^high/ CD24^low)^FA cells were prepared as previously described[36 (link)]. MEGM and MEBM media, EGM media, and Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Lonza. The [E]- and [Z]-4EGI-1were either self-synthesized or ordered from Speed Chemical. 4EGI-N (410E) was self-synthesized. All compounds were dissolved in DMSO. NOD-SCID (strain name: NOD.CB17-Prkdcscid/J) mice were ordered from The Jackson Laboratory. Anti-Ki-67(SP6) antibody was ordered from Vector (^#VP-RM04). Anti-cleaved CASP3 (5A1E) antibody for immunostaining was ordered from Cell Signaling (^#9664s). Anti-CD31 (mouse) antibody was ordered from Dako (^#N1596). Anti-c-Myc (N-term) antibody used for immunostaining was ordered from Epitomics (^#1472-1). Anti-cyclin D1 antibody used for immunostaining was ordered from Neomarkers (^#RM-9104-S). Anti-eIF4E1, anti-eIF4G1, anti-4E-BP1, and anti-β-actin antibodies were ordered from Cell Signaling. Cancer cell lines of SKBR-3, MCF-7, MDA-MB-231 were ordered from ATCC. Anti-c-MYC and anti-cyclin D1 antibodies were ordered from Cell Signaling.

Yi T., Kabha E., Papadopoulos E, & Wagner G. (2014). 4EGI-1 targets breast cancer stem cells by selective inhibition of translation that persists in CSC maintenance, proliferation and metastasis. Oncotarget, 5(15), 6028-6037.

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In Vitro Blood-Brain Barrier Model

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Astrocytes were co-cultured with human umbilical vein endothelial cells (HUVEC) cells according to methods reported in literature [33 (link)]. HUVEC were purchased from ATCC^®. Cells were cultured in EGM Media (Lonza, Basel, Switzerland) supplemented with 10% FBS (Sigma-Aldrich, Milan, Italy), 1% penicillin/streptomycin (Sigma-Aldrich, Milan, Italy) and 2 mM Glutamine (Sigma-Aldrich, Milan, Italy) at 37 °C in a humidified atmosphere of 95% air, 5% CO2. In brief to create the BBB barrier, 4 × 104 astrocytes/cm² were plated on the basolateral side of the flipped 6.5 mm Transwells with polyester membrane with 0.4 μm pore size (Corning Costar, Sigma-Aldrich, Milan, Italy) and left to attach for 4 h. Transwells were then placed into the normal orientation and the cells left to grow for 48 h. After this time, 1 × 105 HUVEC cells/cm² were plated in the apical compartment. The inserts were then placed in a 24-well plate. After 7 days of culture, the Transwells were treated and permeability studies were performed [34 (link)]. To understand the ability of tested substances to cross the blood–brain barrier the medium at the bottom side of the Transwells was quantified over time by measuring the volume and the concentration of BDNF.

Molinari C., Morsanuto V., Ruga S., Notte F., Farghali M., Galla R, & Uberti F. (2020). The Role of BDNF on Aging-Modulation Markers. Brain Sciences, 10(5), 285.

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HUVEC Viability on Stents

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To analyze cell viability, human umbilical vein endothelial cells (HUVECs) in EGM Media (Lonza) were seeded on stents at a density of 50,000 cells/cm². After 7 days, the cells were stained with Live/Dead viability assay kit (Molecular probes) and visualized using an epifluorescence microscope.

Andukuri A., Min I., Hwang P., Alexander G., Marshall L.E., Berry J.L., Wick T.M., Joung Y.K., Yoon Y.S., Brott B.C., Han D.K, & Jun H.W. (2014). Evaluation of the effect of expansion and shear stress on a self-assembled endothelium mimicking nanomatrix coating for drug eluting stents in vitro and in vivo. Biofabrication, 6(3), 035019.

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Culture and Characterization of Endothelial Cells

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VE-cadherin null embryoid body-derived endothelial cells (VEcad^-/-) were maintained in DMEM, 20% FBS, 150mg/L ECGS, 100mg/L heparin, 1× Pen/Strep. HUVECs were obtained from the Yale tissue culture core, cultured in M199, 20% FBS, 150mg/L ECGS, 100mg/L heparin, 1× Pen/Strep, and used between passages 1-8. For FRET experiments, HUVEC were cultured and sheared in EGM media (Lonza). Figure 1B, 3A, Supp 1A and 1B, used bovine aortic endothelial cells (BAECs) obtained from the Coriell Institute and were maintained in DMEM, 20% FBS, 150mg/L ECGS, 100mg/L heparin, 1× Pen/Strep. Measurements were at 2 min because the change in FRET is maximal at that time [5 (link)].

Conway D.E., Coon B.G., Budatha M., Arsenovic P.T., Orsenigo F., Wessel F., Zhang J., Zhuang Z.W., Dejana E., Vestweber D, & Schwartz M.A. (2017). VE-cadherin phosphorylation regulates endothelial fluid shear stress responses through the polarity protein LGN. Current biology : CB, 27(14), 2219-2225.e5.

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Isolation and Characterization of Porcine Aortic Endothelial Cells

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Porcine aortic endothelial cells (PAECs) were isolated from GTKO A- and O-type blood group pig aortas as previously described 18 (link) and cultured in EGM media (Lonza Inc., Allendale, NJ, USA). Porcine peripheral blood mononuclear cells (PBMCs) were isolated from GTKO pig blood by density gradient centrifugation using Ficoll–Hypaque. Human embryonic kidney 293 cells (HEK) and HEK cells expressing the porcine B4GALNT2 cDNA were grown in DMEM media with 10% FBS at 37 °C in a 5% CO₂ incubator. The full length pig B4GALNT2 open reading frame was amplified from the original library isolated clone using a primers set containing a Kozak consensus sequence and in-frame translation stop signals, respectively (Forward: 5′- ACCATGACTTCGTACAGCCCTAG-3′, Reverse: 5′- CAGATACCTTAGGTGGCACATTGGAG-3′). The PCR product was inserted into pcDNA3.1/V5-His TOPO expression vector (Life Technologies, Paisley, UK) and transfected into HEK cells using Lipofectamine-2000 (Life Technologies). A stable G418 resistant HEK clone expressing the porcine B4GALNT2 genes (HEK-B4T) was established and used for further real-time RT-PCR, immunohistochemical staining, complement dependent cytotoxicity, and flow cytometry analysis.

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