Egm 2 bulletkit medium

Manufactured by Lonza

Sourced in Switzerland, United States, United Kingdom, Japan

EGM-2 BulletKit medium is a cell culture medium designed to support the growth and maintenance of endothelial cells. The medium provides the necessary components for endothelial cell proliferation and differentiation, including growth factors, vitamins, and other essential nutrients.

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59 protocols using egm 2 bulletkit medium

Co-culture Angiogenesis Assay for RMST Expression

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A co-culture angiogenesis assay (Hetheridge et al., 2011 (link)) was used to assess RMST expression during EC differentiation. In brief, GFP-HUVECs were cultured in Lonza EGM™-2 BulletKit™ medium supplemented with 2% FBS, SingleQuots™ supplements and gentamicin. Human lung fibroblasts were grown on 10-cm culture plates and maintained in DMEM medium supplemented with 10% FBS. When fibroblasts reached full confluence, 500,000 GFP-HUVECs were added on top of them, and the medium was changed to Lonza EGM™-2 BulletKit™ medium. Medium was changed every other day. After 7days of co-culture, cells were detached with trypsin and the GFP-HUVECs were sorted out using fluorescence-activated cell sorting (FACS) and a BD™ FACSAria Fusion cell sorter located at the Immunophenotyping Platform of the Research Institute of the McGill University Health Centre. As a control, GFP-HUVEC/fibroblast co-culture was maintained for 1day prior to cell sorting. Total RNA was extracted from sorted GFP-HUVECs, and gene expressions of RMST isoforms were measured using qPCR as described above.

Alaqeeli M., Mayaki D, & Hussain S.N. (2021). Long Non-coding RNA Rhabdomyosarcoma 2-Associated Transcript Regulates Angiogenesis in Endothelial Cells. Frontiers in Physiology, 12, 729157.

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Porcine Aortic Endothelial Cell Culture

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Porcine Aortic Endothelial (PAE) cells stably expressing VEGFR2 and Neuropilin-1 (PAE-VEGFR2-NRP1^{12 (link)}), and HEK293T cells were cultured in DMEM with high glucose (No. 1-26F03-I, BioConcept) supplemented with 10% fetal bovine serum (FBS) and Penicillin/ Streptomycin. Human umbilical vein endothelial cells (HUVEC, Life Technologies) were grown in EGM-2 BulletKit medium (Lonza) containing fetal calf serum, hydrocortisone, hFGF-2, VEGF, R3-IGF-1, ascorbic acid, hEGF, GA-1000 and heparin. Cells were propagated in a humidified atmosphere at 37 °C and 5% CO₂.

Xie Y., Mansouri M., Rizk A, & Berger P. (2019). Regulation of VEGFR2 trafficking and signaling by Rab GTPase-activating proteins. Scientific Reports, 9, 13342.

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Fibroblast Ciliogenesis Induction Protocol

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Fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) or EGM-2 Bulletkit medium (Lonza, CC-3162) supplemented with 12% FBS, and passaged onto glass coverslips in 24-well plates. Ciliogenesis was induced by growing cells in medium with reduced serum (0.2%) for 24 hours. Cells were rinsed with phosphate-buffered saline (PBS) once and then fixed in either 100% ice-cold methanol or 4% paraformaldehyde in PBS. Cells were washed in PBS three times, then stored at 4 °C until use. Normal human juvenile foreskin-derived dermal fibroblasts (NHDF) were used as controls (PromoCell: C-12300); in the absence of a normal fetal fibroblast control, equivalent ciliogenesis was confirmed in NHDF and a fibroblast line derived from a 14-week fetus with a non-ciliopathy disease (fetal akinesia). All analyses were performed in cells maintained at low passage numbers.

Cortés C.R., McInerney-Leo A.M., Vogel I., Rondón Galeano M.C., Leo P.J., Harris J.E., Anderson L.K., Keith P.A., Brown M.A., Ramsing M., Duncan E.L., Zankl A, & Wicking C. (2016). Mutations in human C2CD3 cause skeletal dysplasia and provide new insights into phenotypic and cellular consequences of altered C2CD3 function. Scientific Reports, 6, 24083.

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Hypoxia Model of HUVECs

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HUVECs were obtained from ATCC (ATCC, Rockville, MD, USA) and cultured in EGM-2 Bulletkit medium (Lonza, Basel, Switzerland) as in our previous report [15 (link)]. The hypoxia model was performed as our previously described one using Xvivo Closed Incubation System (Xvivo system 300C, BioSpherix, Lacona, New York, USA) [15 (link)]. Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt), a ROS scavenger, was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in DMSO. Rapamycin and 3-methyladenine (3-MA) were obtained from Sigma and dissolved in DMSO.

Tang X., Lin C., Guo D., Qian R., Li X., Shi Z., Liu J., Li X, & Fan L. (2016). CLOCK Promotes Endothelial Damage by Inducing Autophagy through Reactive Oxygen Species. Oxidative Medicine and Cellular Longevity, 2016, 9591482.

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Regulation of SMAD4 and ALK1 in HUVECs

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HUVECs were obtained from the Yale University Vascular Biology and Therapeutics Core Facility and cultured in EGM2-Bullet kit medium (CC-3156 & CC-4176, Lonza). Depletion of SMAD4 and ALK1 was achieved by transfecting 25 pmol of siRNA against SMAD4 (Qiagen, mixture of 4 siRNAs: SI03089527, SI03042508, SI00076041, SI00076020) or against ALK1 (Qiagen, mixture of 2 siRNAs: S102659972 and S102758392) using Lipofectamine RNAiMax (Invitrogen). Transfection efficiency was assessed by western blotting and qPCR. Experiments were performed 60 hours post transfection and results were compared with siRNA CTRL (ON-TARGETplus Non-Targeting Pool D-001810–10-05). Cells were starved for 16 hours in serum free medium and then treated with 10ng.ml⁻¹ of BMP9 for 2 hours.
HUVECs in complete medium were treated for 2 hours with 10μg/ml TBCA or CX-4945 and the effect on PTEN and AKT phosphorylation were detected by Western Blotting.
To determine the half-life of SMAD4 and ALK1, HUVECs were starved in EBM2 and treated with 100μg/ml of cycloheximide for the timepoints indicated.

Ola R., Künzel S.H., Zhang F., Genet G., Chakraborty R., Pibouin-Fragner L., Martin K., Sessa W., Dubrac A, & Eichmann A. (2018). SMAD4 prevents flow induced arterial-venous malformations by inhibiting Casein Kinase 2. Circulation, 138(21), 2379-2394.

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Cell Culture Conditions for Breast Cancer and Endothelial Cell Lines

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MDA-MB 468 cells were cultured in Dulbecco’s Modified Eagle’s Medium High Glucose (DMEM), supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (50 UI/mL) and streptomycin (50 mg/mL). HCC1937 cells were cultured in RPMI 1640 medium, supplemented with 10% FBS, L-glutamine (2 mM), penicillin (50 UI/mL) and streptomycin (50 mg/mL). MDA-MB 231 cells were cultured in MEM Medium, supplemented with 10% FBS, L-glutamine (2 mM), penicillin (50 UI/mL) and streptomycin (50 mg/mL), while HUVEC were cultured in EGM™-2 Bullet kit medium (Lonza). All cell lines grew at 37 °C and 5% CO₂ in a humidified atmosphere and were subcultured prior to confluence using trypsin/EDTA. Cell culture medium and chemicals for cell culture were purchased from Euroclone.

Mazzucchelli S., Truffi M., Baccarini F., Beretta M., Sorrentino L., Bellini M., Rizzuto M.A., Ottria R., Ravelli A., Ciuffreda P., Prosperi D, & Corsi F. (2017). H-Ferritin-nanocaged olaparib: a promising choice for both BRCA-mutated and sporadic triple negative breast cancer. Scientific Reports, 7, 7505.

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HUVEC and hADSC Culture Conditions

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HUVECs (Lonza, Basel, Switzerland) were cultured in EGM-2 BulletKit Medium (Lonza, Basel, Switzerland). The medium supplements contained 2% bovine serum albumin (BSA), hFBF-B, VEGF, R3–insulin-like growth factor-1, ascorbic acid, heparin, fetal bovine serum, hEGF, and GA-1000. hADSCs were cultured in ADSC Growth Medium (Lonza, Basel, Switzerland). For the rest of the assays (unless otherwise stated), Dulbecco’s modified Eagle’s medium (DMEM) (11054020, Thermo Fisher Scientific Inc., USA) with 10% fetal bovine serum (26140079; Thermo Fisher Scientific Inc., USA) was used to exclude the influence from growth factors. Growth medium was changed every other day, and cells were passaged every 6 days. All experiments were conducted using both passage 4 (P4) HUVECs and hADSCs.

Jia J., Jeon E.J., Li M., Richards D.J., Lee S., Jung Y., Barrs R.W., Coyle R., Li X., Chou J.C., Yost M.J., Gerecht S., Cho S.W, & Mei Y. (2020). Evolutionarily conserved sequence motif analysis guides development of chemically defined hydrogels for therapeutic vascularization. Science Advances, 6(28), eaaz5894.

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Generation and Characterization of HCMV-GFP Virus

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HCMV-GFP encoding a green fluorescent protein under the control of the major immediate early promoter was generated on the backbone of the endotheliotropic BAC-cloned TB40/E strain [22,61,62] and prepared as described previously [15]. The HCMV variant RV-TB40-BAC_KL7-SE-EGFP is a repaired version of the TB40/E BAC virus, which contains an intact US region, a self-excisable BAC cassette, and an EGFP reporter under the control of the viral major immediate early promoter [37]. Human retinal pigment epithelial cells (RPE cells) and human fibroblasts (MRC-5 cells) were maintained in DMEM medium supplemented with 10% fetal calf serum. Human umbilical vein endothelial cells (HUVEC) were maintained in EGM-2 Bullet kit medium (Lonza, cc-3162).

Becker J., Kinast V., Döring M., Lipps C., Duran V., Spanier J., Tegtmeyer P.K., Wirth D., Cicin-Sain L., Alcamí A, & Kalinke U. (2018). Human monocyte-derived macrophages inhibit HCMV spread independent of classical antiviral cytokines. Virulence, 9(1), 1669-1684.

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Primary Retinal Endothelial Cell Culture

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Primary human retinal endothelial cells (HRECs) P3 were purchased from Cell Systems (ACBRI 181). HRECs were used within passages five through ten. HRECs were cultured on dishes pre-coated with 0.2% gelatin in phosphate-buffered saline (PBS) for 30 min at 37 °C in EGM-2 BulletKit medium (Lonza, Basel, Switzerland, #CC-3162) supplemented with 2% fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA, USA), 2 mM l-glutamine (Lonza, #17-605E), and 100 U/mL penicillin–100 μg/mL streptomycin (Lonza, #17-602E). Cells were maintained at 37 °C with 5% CO₂.

Hu Z., Cano I., Saez-Torres K.L., LeBlanc M.E., Saint-Geniez M., Ng Y.S., Argüeso P, & D’Amore P.A. (2020). Elements of the Endomucin Extracellular Domain Essential for VEGF-Induced VEGFR2 Activity. Cells, 9(6), 1413.

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Culturing HUVECs for Experiments

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HUVECs were obtained from ATCC and maintained until passage six in EGM-2 Bulletkit medium (Lonza). Cells were split either into 6-well plates or 10 cm dishes and allowed to grow to 70–80% confluence prior to the start of the experiments.

Janaszak-Jasiecka A., Bartoszewska S., Kochan K., Piotrowski A., Kalinowski L., Kamysz W., Ochocka R.J., Bartoszewski R, & Collawn J.F. (2016). miR-429 regulates the transition between Hypoxia-Inducible Factor (HIF)1A and HIF3A expression in human endothelial cells. Scientific Reports, 6, 22775.

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