Endothelial cell growth supplement (ecgs)

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Endothelial cell growth supplement is a laboratory reagent designed to support the growth and proliferation of endothelial cells in cell culture systems. It provides essential nutrients and growth factors required for the maintenance and expansion of endothelial cell lines.

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Endothelial Cell Growth Supplement (ECGS, (2 citations) Endothelial growth supplement (2 citations) Growth supplement (2 citations)

76 protocols using endothelial cell growth supplement (ecgs)

Isolation and Culture of Cardiac Myocytes from SHR Rats

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Cardiac myocytes were isolated from hearts of 7 ‐month ‐old (SHR young) and 14 ‐month ‐old male SHR (old). Briefly, rats were sacrificed under anaesthetic by ketamine i.p., rat hearts were rinsed with Ringer Solution, and the left ventricle was digested mechanically. After a preplating procedure for 60 min to eliminate fibroblasts, the supernatant was transferred to a six‐well culture plate (Corning, USA), and cells were cultivated in Dulbecco's modified Eagle's medium (DMEM, Invitrogen™, UK) with 10% fetal calf serum (FCS, PAA, Austria), ascorbic acid (5 mg/mL, Sigma, USA), transferrin (10 µg/mL, Sigma), NEAA (Non‐Essential Amino Acids, 10 mM, Sigma), sodium‐selenite (20 µg/mL, Sigma, USA), insulin (10 µg/mL, Sigma), Endothelial Cell Growth Supplement (50 µg/mL, Becton Dickinson, Austria), and penicillin/streptomycin (100 U/mL‐100 µg/mL, Gibco, UK). Medium was replaced once a week.
As control H9c2 cells (CRL‐1446, ATCC, USA) were cultivated in DMEM supplemented with 10% FCS, and penicillin/streptomycin (100 U/mL–100 µg/mL). Cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Kapeller B., Mueller J., Losert U., Podesser B.K, & Macfelda K. (2016). Microcurrent stimulation promotes reverse remodelling in cardiomyocytes. ESC Heart Failure, 3(2), 122-130.

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Culturing U937 Monocytes and HUVECs

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The present study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki with the approval of the Institutional Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. The cell lines of U937 monocytes and HUVECs are both purchased from the American Type Culture Collection (Manassas, VA, USA). U937 monocytes are cultured in RPMI1640 medium (Invitrogen, Carlsbad, CA, USA) with 20% fetal bovine serum (Invitrogen). HUVECs are cultured with human endothelial SFM (Invitrogen) with 20% fetal bovine serum (Invitrogen), 100 μg/ml heparin (Sigma-Aldrich, St. Louis, MO, USA), and 200 μg/ml endothelial cell growth supplement (Becton, Dickinson and Company, Frankin Lakes, NJ, USA). The 3 to 5 passages are used for experiments. Both U937 monocytes and HUVECs are cultured in a 5% CO₂ incubator at 37°C and 90% humidity (Thermo Fisher Scientific, Waltham, MA, USA).

Chai Y., Cao Z., Yu R., Liu Y., Yuan D, & Lei L. (2020). Dexmedetomidine Attenuates LPS-Induced Monocyte-Endothelial Adherence via Inhibiting Cx43/PKC-α/NOX2/ROS Signaling Pathway in Monocytes. Oxidative Medicine and Cellular Longevity, 2020, 2930463.

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Isolation of Aortic Endothelial Cells

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Porcine aortic endothelial cells were isolated from GalTKO.hCD46.Neu5GcKO experiments (n=4) and GalTKO.hCD46 historical controls (n=3) via scraping and plated on 0.5% gelatin coated culture flasks (Sigma-Aldrich, St. Louis, MO, USA). Culture media consisted of DMEM (Gibco-Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Atlanta Biological, Lawrenceville, GA, USA), gentamicin, amphotericin B, and endothelial cell growth supplement (Becton Dickinson, San Jose, CA, USA). Human aortic endothelial cells (HAECs) were purchased from Lonza (Allendale, NJ, USA) and cultured in presence of Medium 200 + LSGS Kit supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA).

Cimeno A., Hassanein W., French B.M., Powell J.M., Burdorf L., Goloubeva O., Cheng X., Parsell D.M., Ramsoondar J., Kuravi K., Vaught T., Uluer M.C., Redding E., O’Neill N., Laird C., Hershfeld A., Tatarov I., Thomas K., Ayares D., Azimzadeh A.M., Pierson RN I.I.I., Barth R.N, & LaMattina J.C. (2017). N-glycolylneuraminic acid knock out reduces erythrocyte sequestration and thromboxane elaboration in an ex vivo pig-to-human xenoperfusion model. Xenotransplantation, 24(6), 10.1111/xen.12339.

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HUVEC Tube Formation Assay

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HUVEC cells, kindly provided by Dr MC Cid (IDIBAPS), were cultured in RPMI 1640 supplemented with 20 % defined bovine calf serum (Thermo Fisher Scientific), 0.2 mg/mL endothelial cell growth supplement (Becton Dickinson), 5 units/mL sodium heparin (EMD Millipore), 2 mM glutamine, 100 units/mL penicillin-streptomycin, 50 μg/mL gentamycin (Life Technologies) and 2.5 μg/mL fungizone (Sigma). Supernatant from primary MCL cells (2×10⁶ cells/mL) was collected after 8 hours of incubation with drugs. Twenty-four-well plates were coated with 300 μL of Matrigel (Becton Dickinson) and allowed to polymerize for 30 minutes at 37ºC. Subsequently, 500 μL of the supernatant of interest and 500 μL of HUVEC cells (0.8×10⁵ cells/mL) in its medium were added into each culture well. After 24 hours of incubation, the number of branch points was quantified as the mean of 5 randomly chosen fields from each well. Images were taken at x40 magnification in a DM IL LED microscope coupled to a DFC295 camera with Leica Application Suite v 3.7 software (Leica).

Rosich L., Montraveta A., Xargay-Torrent S., López-Guerra M., Roldán J., Aymerich M., Salaverria I., Beà S., Campo E., Pérez-Galán P., Roué G, & Colomer D. (2014). Dual PI3K/mTOR inhibition is required to effectively impair microenvironment survival signals in mantle cell lymphoma. Oncotarget, 5(16), 6788-6800.

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Endothelial Cells and Monocyte Co-culture

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The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University.
The human umbilical vein endothelial cells (HUVECs) and U937 monocytes used in this study were both purchased from American Type Culture Collection (Manassas, VA, USA). HUVECs were cultured in human endothelial serum-free medium (Invitrogen, Carlsbad, CA, USA) with 20% fetal bovine serum (Invitrogen), 100 U/ml penicillin-streptomycin (Invitrogen), 100 μg/ml heparin (Sigma-Aldrich, St. Louis, MO, USA), and 150 μg/ml endothelial cell growth supplement (Becton, Dickinson and Company, Frankin Lakes, NJ, USA). U937 monocytes were cultured in RPMI1640 medium (Invitrogen), which contains 20% fetal bovine serum (Invitrogen) and 100 U/ml penicillin-streptomycin (Invitrogen). HUVECs and U937 monocytes were both cultured in an incubator (5% CO₂, 37 °C, and 90% humidity) (Thermo Fisher Scientific, Waltham, MA, USA).

Yuan D., Zou Z., Li X., Cheng N., Guo N., Sun G, & Liu D. (2021). A new side-effect of sufentanil: increased monocyte-endothelial adhesion. BMC Anesthesiology, 21, 267.

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Monocyte-Endothelial Cell Co-Culture Protocol

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The present study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki with the approval of the Institutional Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. Both U937 monocytes and HUVECs are obtained from American Type Culture Collection (Manassas, VA, USA). HUVECs are cultured with human endothelial SFM (Invitrogen, Carlsbad, CA, USA), containing 20% fetal bovine serum (Invitrogen), 100 U/ml penicillin-streptomycin (Invitrogen), 100 μg/ml heparin (Sigma-Aldrich, St. Louis, MO, USA), and 150 μg/ml endothelial cell growth supplement (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). U937 monocytes are cultured in RPMI1640 medium (Invitrogen), containing 20% fetal bovine serum (Invitrogen) and 100 U/ml penicillin-streptomycin (Invitrogen). These two cell lines are both cultured in a 5% CO₂ incubator (37°C and 90% humidity) (Thermo Fisher Scientific, Waltham, MA, USA).

Chai Y., Yu R., Liu Y., Wang S., Yuan D, & Chen J. (2020). Dexmedetomidine Attenuates Monocyte-Endothelial Adherence via Inhibiting Connexin43 on Vascular Endothelial Cells. Mediators of Inflammation, 2020, 7039854.

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Culturing Human Aortic Endothelial Cells

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The HAECs were obtained from Lonza (CC2535) and cultured in M199 medium (Hyclone Laboratories) supplemented with 20% FBS (HyClone), endothelial cell growth supplement (ECGS; BD Biosciences), heparin (MilliporeSigma) and 1% penicillin, streptomycin, and amphotericin (PSA; Invitrogen).

Saaoud F., Liu L., Xu K., Cueto R., Shao Y., Lu Y., Sun Y., Snyder N.W., Wu S., Yang L., Zhou Y., Williams D.L., Li C., Martinez L., Vazquez-Padron R.I., Zhao H., Jiang X., Wang H, & Yang X. (2023). Aorta- and liver-generated TMAO enhances trained immunity for increased inflammation via ER stress/mitochondrial ROS/glycolysis pathways. JCI Insight, 8(1), e158183.

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Culturing Human Aortic Endothelial Cells

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HAECs (Lonza, CC2535; Walkersville, MD) were cultured in the M199 medium (Hyclone laboratories, Logan, UT) supplemented with 15% fetal bovine serum (FBS; HyClone, Logan, UT); endothelial cell growth supplement (ECGS, 50 μg/mL); BD Biosciences, San Jose, CA); heparin (50 μg/mL); and 1% penicillin, streptomycin, and amphotericin (PSA; Invitrogen, Carlsbad, CA). HAECs were grown on 0.2% gelatin-coated flasks, plates, or dishes and experiments were performed at passage 9.

Li X., Fang P., Sun Y., Shao Y., Yang W.Y., Jiang X., Wang H, & Yang X. (2019). Anti-inflammatory cytokines IL-35 and IL-10 block atherogenic lysophosphatidylcholine-induced, mitochondrial ROS-mediated innate immune activation, but spare innate immune memory signature in endothelial cells. Redox Biology, 28, 101373.

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Immortalization of Peripheral Nerve Microvascular Endothelial Cells

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Unless otherwise specified, cell-culture reagents were obtained from Life Technologies (Grand Island, NY). An immortalized cell line of peripheral nerve microvascular endoneurial endothelial cells (PNMECs) was prepared from purified rat primary PNMECs using a replication-deficient SV40 retrovirus encoding a temperature sensitive, non-SV40-origin binding mutant of the large T antigen and a selectable neomycin resistance gene as previously described (Langert et al. 2013a ). Immortalized PNMEC cultures were maintained in Ham's F10 basal media supplemented with 10% FBS, 5.6 μg/ml amphotericin B, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml ECGS (BD Biosciences, San Jose, CA), and 0.4 μg/ml heparin (Sigma-Aldrich), under an atmosphere of 5% CO₂/95% air.

Langert K.A., Goshu B, & Stubbs EB J.r. (2016). Attenuation of Experimental Autoimmune Neuritis with Locally Administered Lovastatin-Encapsulating PLGA Nanoparticles. Journal of neurochemistry, 140(2), 334-346.

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Endothelial Cell Stimulation by Peptides

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Custom-made T3 (69–88) and LF-15 (74–88) peptides were obtained from GL Biochem Ltd, Shanghai, China, and T7 peptides were obtained from Proteomics International Pty Ltd, Perth, Australia (see figure 1). Endothelial cells were seeded into 96 well plates at a density of 1×10⁴ cells/cm² in HAMS Nutrient F-12 (JRH Biosciences) containing 20% FBS and 35µg/ml ECGS (BD Biosciences) and grown for a period of 24 hours at 37°C in 5% CO₂. Cells were then quiesced in F12 supplemented with 0.1% BSA for 24 hours. Cells were then treated with 4.5µM LF-15, T3 or T7 peptides or vehicle control (acetonitrile and TFA) in growth medium and incubated at 37°C in 5% CO₂. Peptides were replenished after 24, 72 and 144 hours.

Grafton K.T., Moir L.M., Black J.L., Hansbro N.G., Hansbro P.M., Burgess J.K, & Oliver B.G. (2014). LF-15 & T7, Synthetic Peptides Derived from Tumstatin, Attenuate Aspects of Airway Remodelling in a Murine Model of Chronic OVA-Induced Allergic Airway Disease. PLoS ONE, 9(1), e85655.

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