Endothelial cell growth supplement (ecgs)

Manufactured by Corning

Sourced in United States

Endothelial cell growth supplement is a laboratory product that provides essential nutrients and growth factors to support the in vitro cultivation and proliferation of endothelial cells.

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31 protocols using endothelial cell growth supplement (ecgs)

Isolation and Culture of HUVECs and THP-1 Cells

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Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords using type I collagenase.32 Cells were grown in F‐12K supplemented with 10% FCS, endothelial cell growth supplement (Corning, Bedford, MA), 0.1 mg/mL of heparin (Sigma‐Aldrich), 100 IU of penicillin, and 100 μg/mL of streptomycin (Corning, Manassas, VA). All experiments were performed using cells pooled from multiple donors, passages 3 to 7. The human acute monocyte leukemia cell line, THP‐1 (ATCC, Manassas, VA), was cultured in RPMI 1640 (Hyclone, Logan, UT) supplemented with 10% FCS, 1 mmol/L of l‐glutamine, 100 IU of penicillin, and 100 μg/mL of streptomycin.

Vogel M.E., Idelman G., Konaniah E.S, & Zucker S.D. (2017). Bilirubin Prevents Atherosclerotic Lesion Formation in Low‐Density Lipoprotein Receptor‐Deficient Mice by Inhibiting Endothelial VCAM‐1 and ICAM‐1 Signaling. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 6(4), e004820.

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Culturing and Maintaining Cancer, Endothelial, and Stem Cells

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293T and CT2A cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, #11995-065) containing 10% fetal bovine serum (FBS; Fisher Scientific, #16140071) and 1:100 antibiotic-antimycotic (Gibco, #15140-122), and were purchased from the American Type Culture Collection (ATCC). For stemness maintenance, CT2A cells were cultured in neural stem cell (NSC) proliferation media (Millipore, #SCM005) containing 20 ng/mL basic fibroblast growth factor (bFGF; PeproTech, #100-18B) and epidermal growth factor (EGF; PeproTech, #AF-100-15). iHUVECs were made by transduction of HUVEC with LSNX-16E6E7, an amphotrophic retrovirus encoding the oncoproteins E6 and E7 of human papillomavirus type 16 (provided by Dr. David Klumpp; Northwestern University, Evanston, IL). iHUVECs display all the known characteristics of primary endothelial cells.^{58 (link),59 (link)} Human primary CD34⁺ cells (#70002.1) were purchased from STEMCELL Technologies. iHUVECs and CD34⁺ cells were cultured in endothelial cell basal medium (Gibco, #11111044) containing 12.5 mg/mL endothelial cell growth supplement (Corning, #356006), 10% FBS and 1:100 antibiotic-antimycotic. Patient-derived GSC272 and mouse QPP7 GSCs were cultured in NSC proliferation media (Millipore Corporation, Billerica, MA) containing 20 ng/mL bFGF and EGF. All cells were maintained at 37°C and 5% CO₂ and were confirmed to be mycoplasma-free.

Billiard J., Koh D.S., Babcock D.F, & Hille B. (1997). Protein kinase C as a signal for exocytosis. Proceedings of the National Academy of Sciences of the United States of America, 94(22), 12192-12197.

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Culturing RAW 264.7 Macrophages and HUVECs

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RAW 264.7 macrophages were purchased from ATCC (Manassas, VA, USA), which were cultured in high glucose DMEM (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies, Carlsbad, CA, USA) and 1% Antibiotic-Antimycotic (Gibco, Life Technologies, Carlsbad, CA, USA). Pooled human umbilical vein endothelial cells (HUVECs) were purchased from Lonza Group (Basel, Switzerland). HUVECs were cultured in DMEM/F-12 medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 20% FBS, 50 mg/L endothelial cell growth supplement (Corning, Bedford, MA, USA), 0.1 g/L heparin sodium salt from porcine intestinal mucosa (Sigma-Aldrich, St. Louis, MO, USA), 1.2 g/L sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), HEPES sodium salt (Sigma-Aldrich, St. Louis, MO, USA), and 1% Antibiotic-Antimycotic. The cells were maintained in a humidified incubator at 37°C containing 5% CO₂. HUVECs used for all the experiments were of passage 4-9. RAW 264.7 cells were used from passage 3 after reviving from frozen cells.

Zhou Q., Xu H., Yu W., Li E, & Wang M. (2019). Anti-Inflammatory Effect of an Apigenin-Maillard Reaction Product in Macrophages and Macrophage-Endothelial Cocultures. Oxidative Medicine and Cellular Longevity, 2019, 9026456.

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Culturing Human Endothelial and Keratinocyte Cells

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Human umbilical vein endothelial cells (HUVECs; American Type Culture Collection (ATCC), Manassas, VA, USA; CRL-1730) were cultured in F-12K (ATCC 30-2204) supplemented with 0.1 mg/mL heparin (#H3393; Sigma-Aldrich Inc., St. Louis, MO, USA), 0.03 mg/mL endothelial cell growth supplement (356006, Corning Inc., New York, NY, USA), and 10% FBS (Corning Inc., New York, NY, USA). Human keratinocytes (HaCaT; AddexBio Technologies, San Diego, CA, USA) were cultured in AddexBio-optimized DMEM (C0003-02) with 10% FBS. Cells were maintained at 37 °C with 5% CO₂.

Curtis T.M., Nilon A.M., Greenberg A.J., Besner M., Scibek J.J., Nichols J.A, & Huie J.L. (2023). Odorant Binding Causes Cytoskeletal Rearrangement, Leading to Detectable Changes in Endothelial and Epithelial Barrier Function and Micromotion. Biosensors, 13(3), 329.

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Culturing Primary Mouse and Human Brain Endothelial Cells

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Primary C57BL/6 mouse brain microvascular endothelial cells (MBECs; C57-6023,
Cell Biologics, Chicago, IL) were seeded in 12-well plates coated with
Gelatin-Based Coating Solution (6950, Cell Biologics) for cell assays and
allowed to grow to 80–90% confluency before treatment. Cells were grown in
Complete Mouse Endothelial Cell Medium with supplemental kit (M1168 + kit, Cell
Biologics), at 37°C in 5% CO₂. All experiments were completed on cell
passage 5.
Human cerebral microvascular endothelial cells were isolated from post-mortem
brains of donors and transfected with plasmid containing SV40 large T antigen
(HBEC-5i; CRL-3245^TM, ATCC®, Manassas, VA). Cells were grown in F12:
DMEM plus 10% FBS and Endothelial Cell Growth Supplement (356,006, Corning) on
0.1% Gelatin (PCS-999–027, ATCC®). To exclude the effect of growth factors,
culture medium was changed to F12:DMEM without serum 4 hours prior to treatment.
All experiments were completed on cell passage 4.
All insulin stimulation and inhibitor treatments were performed at 37°C.
Hyperinsulinemic conditioning was induced in MBECs by incubating cells for
12 hours in 20 nM human recombinant insulin (I9278, Sigma, St. Louis, MO), and
HBEC-5is in 5 nM human recombinant insulin for 12 hours. Time and concentrations
for hyperinsulinemic conditioning were determined in pilot studies, data not
presented here.

Watson L.S., Wilken-Resman B., Williams A., DiLucia S., Sanchez G., McLeod T.L, & Sims-Robinson C. (2022). Hyperinsulinemia alters insulin receptor presentation and internalization in brain microvascular endothelial cells. Diabetes & Vascular Disease Research, 19(4), 14791641221118626.

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Culturing Diverse Cell Lines for Experiments

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HEK293T, MCF7, and C2C12 cells were grown in DMEM (Biochrom AG) supplemented with 10% fetal calf serum (FCS; Biochrom AG), 2 mM l-glutamine, and penicillin (100 U/ml)/streptomycin (10 µg/ml) (PAA Laboratories) at 37°C and 10% (C2C12) or 5% CO₂. Immortalized human myoblasts were cultured in skeletal muscle growth medium (Provitro) supplemented with supplement mix (Provitro), 50 ng/ml amphotericin, 50 µg/ml gentamicin, 10% FCS, 2 mM l-glutamine, and penicillin (100 U/ml)/streptomycin (10 µg/ml) at 37°C and 5% CO₂. hFOBs (1.19) were cultured in a 1:1 mixture of DMEM and Ham’s F12 medium supplemented with 10% FCS, 2 mM l-glutamine, penicillin (100 U/ml)/streptomycin (10 µg/ml), and 0.3 mg/ml G418 (Biochrom AG) at 34°C with 5% CO₂ to keep them in a proliferative state. HUVECs were a kind gift from M. Lorenz and V. Stangl (Charité Universitätsmedizin, Berlin, Germany) and cultured on gelatin-coated tissue culture ware in M199 medium supplemented with 20% FCS, 50 µg/ml endothelial cell growth supplement (Corning), 25 µg/ml heparin, 2 mM l-glutamine, and penicillin (100 U/ml)/streptomycin (10 µg/ml) at 37°C and 5% CO₂. HUVECs were used at passage 3 in all experiments. Unless stated otherwise, all cells were starved for 5 h prior to stimulation with their respective growth medium, without FCS supplement, containing 2 mM l-glutamine and penicillin/streptomycin.

Brunner P., Hastar N., Kaehler C., Burdzinski W., Jatzlau J, & Knaus P. (2020). AMOT130 drives BMP-SMAD signaling at the apical membrane in polarized cells. Molecular Biology of the Cell, 31(2), 118-130.

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Human Umbilical Vein Endothelial Cell Culture

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Cell culture reagents were obtained from Mediatech (Manassas, VA) unless otherwise specified. Immortalized human umbilical vein endothelial cells (iHUVEC, generated by transducing HUVECs with the recombinant retrovirus LXSN16 E6/E7) and PEC02 cells (generated by transducing iHUVECs with a lentivirus expressing a PECAM1-specific siRNA PEC02 - (Privratsky et al., 2011 (link))) were maintained in RPMI1640 medium (Mediatech), 10% FBS (Sigma, St Louis, MO), 5% human AB serum (Gemini, West Sacramento, CA), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.5 mg/ml endothelial cell growth supplement (Corning, Corning, NY) as previously described (Privratsky et al., 2011 (link)).

Liao D., Mei H., Hu Y., Newman D.K, & Newman P.J. (2017). CRISPR-mediated deletion of the PECAM-1 cytoplasmic domain increases receptor lateral mobility and strengthens endothelial cell junctional integrity. Life sciences, 193, 186-193.

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Isolation and Characterization of Brain Endothelial Cells

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Three preparations of BECs were used: commercially available rat brain microvascular endothelial cells (RBMVECs; Cell Applications, San Diego, CA, USA), freshly isolated rat BECs (iBECs) and human BECs (hBECs). Isolated astrocytes were employed in a transwell co-culture system.
RBMVECs, passages 5–10, were cultured as per the manufacturer’s recommendations. iBECs were isolated from Sprague Dawley rats using a previously established method [27 ] (see ESM Methods ‘Harvest of iBECs’). iBECs were 98% CD31+ by flow cytometry, expressed claudin-5, and had increased phosphorylated Akt in response to insulin stimulation (see ESM Methods ‘Flow cytometry’ and ‘Immunofluorescence’, ESM Fig. 1). hBECs were a gift from J. Catravas and were 99% positive for DiI-Ac-LDL (acetylated Dil-labelled LDL) uptake and endothelial nitric oxide synthase (eNOS) expression [28 (link)]. hBECs were cultured in DMEM/Ham’s F12 medium (F12) with HEPES and L-glutamine, 10% FBS, endothelial cell growth supplement (Corning, Corning, NY, USA), heparin (Sigma-Aldrich, St Louis, MO, USA) and Anti-Anti [29 (link)]. Astrocytes were isolated from Sprague Dawley rat pups at postnatal day 3–10 as per established protocols [30 ]. Astrocytes were positive for GFAP (glial fibrillary acidic protein) by immunofluorescence (see ESM Methods ‘Immunofluorescence’, ESM Fig. 2) and western blot.

Gray S.M., Aylor K.W, & Barrett E.J. (2017). Unravelling the regulation of insulin transport across the brain endothelial cell. Diabetologia, 60(8), 1512-1521.

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Isolation and Culture of Aortic Endothelial Cells

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Aortas were cut open lengthwise to expose the endothelial surface. Vessels were incubated in a collagenase II (Worthington) solution (2 mg/ml in DMEM) at 37°C with the endothelial surface facing down for 30 min. Collagenase was blocked 1:1 with complete endothelial cell growth medium [DMEM supplemented with 10% fetal bovine serum, 1% Antibiotic-Antimycotic solution (Gibco, MA), 4 μg/ml endothelial cell growth supplement (Corning, 354006), 1% Non-essential amino acids (Gibco, MA), and 10 mM HEPES (Gibco, MA)]. The endothelial surface of each vessel was scraped into fibronectin-coated tissue culture dishes containing complete medium. Cultures were expanded and frozen at passage 1. The endothelial phenotype of the preparation was confirmed by evaluating cellular uptake of the endothelium-specific marker DiI-acetylated low-density lipoprotein. Experiments were conducted in cells obtained from three control and three diabetic male and four control and four diabetic female rats. The day before the experiments, cells were plated in commercial endothelial cell growth medium (ScienCell, CA) supplemented with 25 mM glucose (ScienCell, CA).

Akther F., Razan M.R., Shaligram S., Graham J.L., Stanhope K.L., Allen K.N., Vázquez-Medina J.P., Havel P.J, & Rahimian R. (2021). Potentiation of Acetylcholine-Induced Relaxation of Aorta in Male UC Davis Type 2 Diabetes Mellitus (UCD-T2DM) Rats: Sex-Specific Responses. Frontiers in Physiology, 12, 616317.

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Isolation and Culture of Endothelial Cells

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Bovine Aortic Endothelial Cells (BAECs) (VEC Technologies, Rensselaer, NY, USA), selected for their ease of transfections and strong migratory responses to VEGF stimulation, were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (HyClone Laboratories), 2.0 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.
Microvascular mouse lung endothelial cells (MLECs) were isolated from the lungs of eNOS^+/+ and eNOS^−/− mice as previously described [27 (link)]. MLEC were grown in DMEM:F12 (1:1) medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), containing 20% FBS (HyClone, GE Healthcare Life Sciences, Piscataway, NJ, USA), 0.05 mg/mL Endothelial cell growth supplement (Corning Life Science, Tewksbury, MA, USA), 0.1 mg/mL heparin (Millipore-Sigma, St-Louis, MO, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific) and plated in 0.1% gelatin-coated T75 flasks. Magnetic Dynabeads (Thermo Fisher Scientific) were conjugated with anti-mouse CD102 (clone 3C4; BD Biosciences, Franklin Lakes, NJ, USA) antibody. Beads were added to each flask and incubated for 1 h at 4 °C. Cells were subsequently selected in a magnetic field for 10 min following trypsinization.

Smith T.L., Oubaha M., Cagnone G., Boscher C., Kim J.S., El Bakkouri Y., Zhang Y., Chidiac R., Corriveau J., Delisle C., Andelfinger G.U., Sapieha P., Joyal J.S, & Gratton J.P. (2021). eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity. Cellular and Molecular Life Sciences, 79(1), 37.

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