Egm 2 mv singlequot kit

Manufactured by Lonza

The EGM-2 MV SingleQuot Kit is a laboratory product designed to support the growth and maintenance of various cell types in cell culture. The kit contains a set of supplementary components that are added to a base medium to create a complete growth environment for cells. The core function of the kit is to provide the necessary nutrients, growth factors, and other additives required for optimal cell growth and proliferation.

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11 protocols using egm 2 mv singlequot kit

Cultivation of Endothelial and Fibroblast Cells

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Human lymphatic endothelial cells isolated from the lymph node (HLECs, 2500, ScienCell, Carlsbad, CA) and human umbilical vein endothelial cells (HUVECs, C2517A, Lonza, Allandale, NJ) were cultured separately in standard cell culture flasks coated with fibronectin (5μg/cm², F1141-5MG, Sigma Aldrich, St. Louis, MO) at a starting cell concentration of 5 x 10⁵ as per supplier instructions. Cultures were maintained with Endothelial Basal Medium-2 (EBM-2, CC-3156) supplemented with EGM-2 MV SingleQuot Kit (CC-4147, Lonza, Allandale, NJ). HLECs and HUVECs were cultured to 95% confluency at passages 4 and 5 for all experiments. Normal mammary fibroblasts (provided by Dr. Lisa Arendt’s lab at UW-Madison) and breast cancer-associated fibroblasts (provided by Dr. Andreas Friedl’s lab at UW-Madison) were cultured separately in standard flasks at a starting cell concentration of 5 x 10⁵. Fibroblasts were maintained with Dulbecco’s Modified Eagle Medium (11965092, ThermoFisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum. All cultures were kept in a humidified incubator at 37 °C with 5% CO₂.

Gong M.M., Lugo-Cintron K.M., White B.R., Kerr S.C., Harari P.M, & Beebe D.J. (2019). Human organotypic lymphatic vessel model elucidates microenvironment-dependent signaling and barrier function. Biomaterials, 214, 119225.

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Inflammatory Triggered EV Isolation

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HUVEC (BD Bioscience, cat # 354151) at passages three to six were seeded at a density of 600,000 cells in EBM-2 (Lonza) supplemented with EGM-2 MV SingleQuot Kit (Lonza) and 5% vesicles-depleted fetal bovine serum (System Bioscience). When HUVEC were grown up to 70–75% confluency, cells were washed twice with HEPES buffer saline (Lonza) and cells were then inflammatory triggered by adding 10 ng/ml TNF-α in refreshed medium for overnight (13 (link)). Afterward, the supernatants were collected for the EV isolation. All collected supernatant samples containing EV were stored at −80°C until EV isolation procedures.
THP-1 (ATCC^® TIB-202™) were grown in RPMI-1640 (Life Technologies) medium supplemented with 10% vesicles-depleted fetal bovine serum (System Bioscience) and 1% penicillin–streptomycin–amphotericin B (Lonza Biowhittaker). All cell lines were incubated in a humidified atmosphere condition of 5% CO₂/95% O₂ at 37°C.

Hosseinkhani B., Kuypers S., van den Akker N.M., Molin D.G, & Michiels L. (2018). Extracellular Vesicles Work as a Functional Inflammatory Mediator Between Vascular Endothelial Cells and Immune Cells. Frontiers in Immunology, 9, 1789.

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Co-culture of Lymphatic Endothelial Cells and Breast Cancer Cells

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Human lymphatic endothelial cells (HLECs, ScienCell, 2500) were cultured in standard cell culture flasks coated with fibronectin (5 µg/cm², Sigma Aldrich, F1141–5MG) at a starting cell concentration of 5·10⁵. Cultures were maintained with endothelial basal medium-2 (Lonza, CC-3156) supplemented with EGM-2 MV SingleQuot Kit (Lonza, CC-4147). HLECs were cultured to 90–95% confluency at passages 3 to 5 for all experiments and 3 different lots of lymphatic endothelial cells were used for the experiments. We used human mammary adenocarcinoma cells, MDA-MB-231, transfected to stably expressing green fluorescent protein (GFP), a kind gift from Dr. Suzanne Ponik (University of Wisconsin, Madison). MDA-MB-231s were routinely cultured in high glucose DMEM (Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS, VWR, 97068–085) and 1% penicillin/streptomycin (ThermoFisher, 15140–122). For all experiments, a one to one mixture of lymphatic endothelial cell and MDA-MB-231 cell media was used (i.e., EGM-2 MV to 10% FBS, 1% P/S high glucose DMEM), called experimental media through the text. All cultures were kept in a humidified incubator at 37°C with 5% CO₂.

Lugo-Cintrón K.M., Ayuso J.M., White B.R., Harari P.M., Ponik S., Beebe D.J., Gong M.M, & Virumbrales-Muñoz M. (2020). Matrix Density Drives 3D Organotypic Lymphatic Vessel Activation in a Microfluidic Model of the Breast Tumor Microenvironment. Lab on a chip, 20(9), 1586-1600.

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Direct Reprogramming of Fibroblasts

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Primary human adult dermal fibroblasts (ATCC PCS-201-012) were purchased, mycoplasma-free and certified, directly from ATCC. No further cell line authentication/testing was conducted. The cells were expanded in fibroblast basal medium supplemented with fibroblast growth kit–serum-free (ATCC PCS 201–040) and penicillin/streptomycin. E12.5–E14 mouse embryonic fibroblasts (MEFs) were cultured in DMEM/F12 supplemented with 10% fetal bovine serum. Non-viral cell transfection and reprogramming experiments were conducted via three-dimensional nanochannel electroporation (NEP), as described previously^{11 (link)}. Briefly, the cells were first grown to full confluency overnight on the 3D NEP device. Subsequently, a pulsed electric field was used to deliver a cocktail of plasmids (0.05 μg μl⁻¹) into the cells consisting of a 1:1:1 mixture of Fli1:Etv2:Foxc2. The cells were then collected 24 h after plasmid delivery, placed in EBM-2 basal medium (CC-3156, Lonza) supplemented with EGM-2 MV SingleQuot kit (CC-4147, Lonza) and further processed for additional experiments/measurements. Etv2 and Fli1 plasmids were donated by A. Ferdous (Department of Internal Medicine, UT Southwestern Medical Center, Texas). Foxc2 plasmids were donated by T. Kume (Department of Medicine–Cardiology and Pharmacology, Northwestern University–FCVRI, Chicago).

Gallego-Perez D., Pal D., Ghatak S., Malkoc V., Higuita-Castro N., Gnyawali S., Chang L., Liao W.C., Shi J., Sinha M., Singh K., Steen E., Sunyecz A., Stewart R., Moore J., Ziebro T., Northcutt R.G., Homsy M., Bertani P., Lu W., Roy S., Khanna S., Rink C., Sundaresan V.B., Otero J.J., Lee L.J, & Sen C.K. (2017). Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue. Nature nanotechnology, 12(10), 974-979.

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Acquisition and culture of endothelial and smooth muscle cells

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We purchased pooled human umbilical vein endothelial cells (HUVEC; C2519A), human aortic endothelial cells (HAEC; CC-2535), and human coronary artery endothelial cells (HCAEC; CC-2585; Lonza) and cultured them in EBM-2 media supplemented with EGM-2 or EGM2-MV SingleQuot kit. We purchased immortalized HAEC (teloHAEC; CRL-4052; ATCC) and cultured them in vascular cell basal medium supplemented with vascular endothelial cell growth kit and puromycin at 0.3 μg/mL. Human coronary artery smooth muscle cells (HCASMC) and human aortic smooth muscle cells (HASMC) were obtained from Dr. Tardif’s lab and cultured in medium 231 (M-231-500) supplemented with smooth muscle growth supplement (S-007-25; Gibco). Monocytes were obtained from Dr. Rioux’s lab and we cultured them in RPMI with 10% fetal bovine serum. RNA from tissues was extracted either with the Ribopure Kit (Ambion) or using EZ1-XL Advance and Qiagen EZ1 RNA Tissue Mini Kit. hCA were obtained from the “Réseau d’Échanges de Tissus et d’Échantillons Biologiques” (RÉTEB) biorepository at the Montreal Heart Institute and Quebec Heart and Lung Institute. We purchased adult brain total RNA (540005–41, lot#6048990) and adult heart total RNA (540011–41, lot#6056165; Agilent Technologies).

Codina-Fauteux V.A., Beaudoin M., Lalonde S., Lo K.S, & Lettre G. (2018). PHACTR1 splicing isoforms and eQTLs in atherosclerosis-relevant human cells. BMC Medical Genetics, 19, 97.

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HUVEC Culture and Inflammatory Induction

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HUVEC (BD Bioscience, cat # 354,151) at passage 3–5 were grown in EBM-2 (Lonza) supplemented with EGM-2 MV SingleQuot Kit (Lonza; except the SingleQuot Kit foetal bovine serum) and 5% exosome-depleted foetal bovine serum (EXO-FBS-250A-1, System Bioscience) up to 70–75% confluency to minimize apoptosis. Confluent cells were rinsed twice with PBS (Lonza) and TNF-α (ImmunoTools, cat: 11,343,015)–based inflammation induction in cells was then performed at a final concentration of 10 ng/mL in a refreshed medium for 24 h [19 ]. Cells were incubated in a humidified atmosphere condition of 5% CO₂/95% O₂ at 37°C and routinely checked to be free of mycoplasma contamination.

Hosseinkhani B., van den Akker N.M., Molin D.G, & Michiels L. (2020). (Sub)populations of extracellular vesicles released by TNF-α –triggered human endothelial cells promote vascular inflammation and monocyte migration. Journal of Extracellular Vesicles, 9(1), 1801153.

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Cell Culture Protocols for Cancer and Lymphatic Endothelial Cells

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MCF7 (ATCC) and MDA-MB-231 (ATCC) cells were cultured in standard cell culture flasks and maintained in RPMI 1640 (BE12–702F, Lonza) supplemented with 10% FBS (97068–085, VWR). Human lymphatic endothelial cells (HLECs) isolated from the lymph node (2500, ScienCell) were cultured in standard cell culture flasks coated with fibronectin (5μg/cm², F1141–5MG, Sigma Aldrich) at a starting cell concentration of 5 × 10⁵ as per supplier instructions. HLECs were maintained with Endothelial Basal Medium-2 (CC-3156, Lonza) supplemented with EGM-2 MV SingleQuot Kit (CC-4147, Lonza) and used at 95% confluency at passages 3 and 4 for all experiments.

Ayuso J.M., Gong M.M., Skala M.C., Harari P.M, & Beebe D.J. (2020). Human tumor-lymphatic microfluidic model reveals differential conditioning of lymphatic vessels by breast cancer cells. Advanced healthcare materials, 9(3), e1900925.

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Nanoelectroporation of Dermal Cells for miRNA Delivery

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Primary human adult dermal fibroblasts (ATCC, Manassas, VA, cat. no. PCS-201-012) were expanded in fibroblast basal medium (ATCC cat. no. PCS-201-030) supplemented with fibroblast growth kit-serum-free (ATCC, cat. no. PCS-201-040) containing Penicillin-Streptomycin (10,000 U/mL) solution (Gibco/Life Technologies, Waltham, MA, cat. no. 15140122) at 37 °C in humidified chamber consisting of 95% air and 5% CO₂. Human dermal microvascular endothelial cells (HMECs)^{50 (link)} were cultured in MCDB-131 medium (Gibco/Life Technologies, cat. no. 10372-019) supplemented with 10% FBS, 10 mM L-glutamine and 100 IU/mL penicillin and 0.1 mg/mL streptomycin.
In vitro nanoelectroporation was conducted via 3D Nanochannel Electroporation (NEP) as described previously^{51 (link)}. Briefly, the cells were first grown to full confluency overnight on the 3D NEP device. Subsequently, a pulsed electric field was used to deliver control or miR-200b inhibitor (50 nM) into the cells. The cells were then harvested 24 h after miRNA delivery, placed in EBM-2 basal medium (Lonza, cat. no. CC-3156) supplemented with EGM-2 MV SingleQuot kit components (Lonza, cat. no. CC-414 Supplementary 7) and processed further for additional experiments.

Pal D., Ghatak S., Singh K., Abouhashem A.S., Kumar M., El Masry M.S., Mohanty S.K., Palakurti R., Rustagi Y., Tabasum S., Khona D.K., Khanna S., Kacar S., Srivastava R., Bhasme P., Verma S.S., Hernandez E., Sharma A., Reese D., Verma P., Ghosh N., Gorain M., Wan J., Liu S., Liu Y., Castro N.H., Gnyawali S.C., Lawrence W., Moore J., Perez D.G., Roy S., Yoder M.C, & Sen C.K. (2023). Identification of a physiologic vasculogenic fibroblast state to achieve tissue repair. Nature Communications, 14, 1129.

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Culturing Human Lymphatic Endothelial Cells

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Human lymphatic endothelial cells (HLECs, ScienCell, 2500) were cultured in standard T75 cm² cell culture flasks (Corning, CLS430641U) coated with fibronectin as previously described [30 ,31 ] at a starting cell concentration of 5·10⁵ cells. Cultures were maintained in endothelial basal medium-2 (Lonza, CC-3156) with EGM-2 MV SingleQuot Kit (Lonza, CC-4147), hereafter referred to as endothelial media. HLECs were cultured to 90-95% confluency at passage 3 for all experiments. Primary fibroblasts were routinely cultured in DMEM (Gibco, 11965092) with 10% fetal bovine serum (FBS, VWR, 97068-085), 1% Pen/Strep (ThermoFisher, 15140-122), and 1 µg/mL hydrocortisone (STEMCELL Technologies Inc., 07925). Primary human normal oral fibroblasts (HOrF, ScienCell, 2640) were cultured in standard T75 cm² cell culture flasks (Corning, CLS430641U) at a starting cell concentration of 5·10⁵ cells in fibroblast media (ScienCell, 2301). All cultures were kept in a humidified incubator at 37°C with 5% CO₂. Media was refreshed every 2-3 days, and cells were cultured to 90-95% confluency.

Lugo-Cintrón K.M., Ayuso J.M., Humayun M., Gong M.M., Kerr S.C., Ponik S.M., Harari P.M., Virumbrales-Muñoz M, & Beebe D.J. (2021). Primary Head and Neck Tumour-Derived Fibroblasts Promote Lymphangiogenesis in a Lymphatic Organotypic Co-culture Model. EBioMedicine, 73, 103634.

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Cell Culture Conditions for Cancer and Endothelial Cells

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UM-SCC-1 and UM-SCC-6 cells were cultured with DMEM high glucose (Gibco, 11964-092) supplemented with Penicillin/Streptomycin (Gibco, 15140) and 10% FBS (VWR, Radnor). Human lymphatic endothelial cells (HLECs, ScienCell, 2500) were cultured with endothelial basal medium-2 (Lonza, CC-3156) supplemented with EGM-2 MV SingleQuot Kit (Lonza, CC-4147). Primary human normal oral fibroblasts (HOrFs, ScienCell, 2640) were cultured with fibroblast media (ScienCell, 2301). Cells were maintained at 37 C with 5% CO2 and media was refreshed every 2-3 days.

Yada R.C., Desa D.E., Gillette A.A., Bartels E., Harari P.M., Skala M.C., Beebe D.J, & Kerr S.C. (2023). Microphysiological head and neck cancer model identifies novel role of lymphatically secreted monocyte migration inhibitory factor in cancer cell migration and metabolism. Biomaterials, 298.

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