Recombinant human epidermal growth factor

Manufactured by R&D Systems

Sourced in United States

Recombinant human epidermal growth factor (EGF) is a protein that plays a crucial role in cellular proliferation, differentiation, and survival. It is a potent mitogen that stimulates the growth and development of various cell types, particularly epithelial cells. This recombinant EGF is produced using a non-human expression system.

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39 protocols using recombinant human epidermal growth factor

HUVEC Isolation and Culture Protocol

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Endothelial cells were harvested from fresh umbilical cords obtained during the normal delivery of healthy neonates by collagenase digestion as described earlier [6 (link)]. HUVECs were grown in gelatin-coated flasks (Corning^® Costar^®, Corning NY, USA) in an MCDB131 medium (ThermoFisher Scientific, Waltham, MA, USA) completed with 5% heat-inactivated calf serum (FCS), 2 ng/mL human recombinant epidermal growth factor (R&D Systems, Minneapolis, MN, USA), 1 ng/mL human recombinant basic fibroblast growth factor, 0.3% insulin transferrin selenium (ThermoFisher Scientific), 1% chemically defined lipid concentrate (ThermoFisher Scientific), 1% Glutamax solution (ThermoFisher Scientific), 1% penicillin–streptomycin antibiotic solution, 5 μg/mL ascorbic acid, 250 nM hydrocortisone, 10 nM HEPES, and 7.5 U/mL heparin. For some experiments, the medium was replaced with AIM-V medium (ThermoFisher Scientific) completed with 1% FCS, 2 ng/mL human recombinant epidermal growth factor (R&D Systems), 1 ng/mL human recombinant basic fibroblast growth factor, and 7.5 U/mL heparin.
All experiments were performed in at least 3 independent, primary HUVEC cultures obtained from different individuals before passage 5.

Németh Z., Demeter F., Dobó J., Gál P, & Cervenak L. (2024). Complement MASP-1 Modifies Endothelial Wound Healing. International Journal of Molecular Sciences, 25(7), 4048.

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Isolation and Culture of Human GBM Cells

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Human patient derived high grade glioma cells were isolated from dissociated surgical tumor specimens with approval of Kobe University Hospital Institutional Review Board [37 (link)]. Human patient derived cells were cultured in EF20 medium composed of Neurobasal medium (ThermoFisher Gibco) supplemented with 3 mM l-Glutamine (Corning), 1 × B27 supplement (ThermoFisher Gibco), 0.5 × N2 supplement (ThermoFisher Gibco), 20 ng/ml recombinant human epidermal growth factor (R&D Systems), 20 ng/ml recombinant human fibroblast growth factor-2 (PeproTech), and 0.5 × penicillin G/streptomycin/amphotericin B complex (Corning) at 37 °C and 5% CO₂.

Tanaka K., Sasayama T., Nagashima H., Irino Y., Takahashi M., Izumi Y., Uno T., Satoh N., Kitta A., Kyotani K., Fujita Y., Hashiguchi M., Nakai T., Kohta M., Uozumi Y., Shinohara M., Hosoda K., Bamba T, & Kohmura E. (2021). Glioma cells require one-carbon metabolism to survive glutamine starvation. Acta Neuropathologica Communications, 9, 16.

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Cultivation and Characterization of Human Mucoepidermoid Carcinoma Cell Lines

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University of Michigan Human Mucoepidermoid Carcinoma cell lines (UM-HMC-1, UM-HMC-3A, and UM-HMC-3B) (RRID:CVCL_Y473, RRID:CVCL_Y471, RRID:CVCL_Y472, respectively) were generated from surgical specimens (23 (link)). These cells were cultured in high glucose Dulbecco’s Modified Eagle’s Medium (Invitrogen; Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Atlanta Biologicals; Flowery Branch, GA, USA), 1% L-Glutamine (MilliporeSigma; Burlington, MA, USA), 1% Antibiotic-Antimycotic (MilliporeSigma), 400 ng/mL hydrocortisone (StemCell Technologies; Vancouver, BC, Canada), 20 ng/mL recombinant human epidermal growth factor (R&D Systems; Minneapolis, MN), and 5 μg/mL recombinant human insulin (Sigma-Aldrich; Burlington, MA, USA) at 37°C and 5% CO₂. Low passage primary human microvascular endothelial cells (HDMEC) (Lonza; Morristown, NJ, USA) were cultured in endothelial growth medium-2 for microvascular cells (Lonza). Small molecule inhibitors of MDM2-p53 interaction (i.e., MI-773, APG-115, and MI-1061) and MDM2 degrader (MD-224) were provided by Shaomeng Wang (University of Michigan) (22 (link), 24 (link), 25 (link)). MG132 (MilliporeSigma) was used to inhibit proteasomal degradation. PTC596 (MedChemExpress; Monmouth Junction, NJ), a small molecule inhibitor of Bmi-1, was used to evaluate the direct impact of Bmi-1 on MEC stemness.

Rodriguez-Ramirez C., Zhang Z., Warner K.A., Herzog A.E., Mantesso A., Zhang Z., Yoon E., Wang S., Wicha M.S, & Nör J.E. (2022). p53 inhibits Bmi-1-driven self-renewal and defines salivary gland cancer stemness. Clinical cancer research : an official journal of the American Association for Cancer Research, 28(21), 4757-4770.

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Culturing Patient-Derived Glioblastoma Stem Cells

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The patient-derived GSCs were well characterized (Table 1) and cultured in NS-A medium (90% NeuroCult NS-A Basal Medium Human plus 10% Human NeuroCult NS-A proliferation Supplements, StemCell Technologies, Vancouver, British Columbia, Canada in 3D sphere as we described before [45 (link)]. Complete medium was supplied with recombinant human epidermal growth factor (R&D System, Minneapolis, MN, USA) and 100 units/mL penicillin-100 μg/mL streptomycin (Life Technologies, Carlsbad, CA, USA). Anti-TUSC3 and anti-MGMT antibodies were obtained from Abcam (Cambridge, United Kingdom); anti-β-actin IgG-HRP was obtained from Santa Cruz Biotech (Dallas, TX, USA) and anti-DNMT1, Goat anti-Rabbit IgG-HRP and Goat anti-mouse IgG-HRP were obtained from CellSignaling Technology (Danvers, MA, USA). Temozolomide (TMZ) was obtained from Sigma (St. Louis, MO, USA). 5-Azacitidine (5-Aza) and Lomeguatrib were obtained from Selleckchem (Houston, TX, USA).

Wu Q., Berglund A.E., Macaulay R.J, & Etame A.B. (2023). Epigenetic Activation of TUSC3 Sensitizes Glioblastoma to Temozolomide Independent of MGMT Promoter Methylation Status. International Journal of Molecular Sciences, 24(20), 15179.

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Glioblastoma Tumorsphere Formation Assay

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GBM cells were plated at a density of 5.0 × 10⁴ cells/ml in stem cell medium containing DMEM-F12 (Mediatech) supplemented with 20 ng/μl recombinant human epidermal growth factor (R&D systems), 10 ng/μl recombinant human basic fibroblast growth factor (R&D systems), 1 × B27 supplement (Gibco) using 100 mm ultra-low attachment culture dishes and maintained in a humidified 37°C, 5% CO₂ incubator as described previously (27 (link)). For the tumorsphere-formation assay, T98G or U87R cells (2.0 × 10⁵ cells/well in six-well plates) were treated with the either scL-siMAL or scL-siCTRL nanocomplexes at a concentration of 100 nM. Twenty-four hours later, cells were collected by trypsin and re-seeded in 96-well plates at a density of 100 cells/well. Cells were further incubated with the stem cell medium. On day 10 of culture, the spheres (>100 μm) were scored using Olympus CK2 microscope.

Kim S.S., Harford J.B., Moghe M., Rait A., Pirollo K.F, & Chang E.H. (2017). Targeted nanocomplex carrying siRNA against MALAT1 sensitizes glioblastoma to temozolomide. Nucleic Acids Research, 46(3), 1424-1440.

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Glioblastoma cell culture protocol

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U251 and U87 human glioblastoma cell lines (ATCC) were cultured in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 units/mL penicillin and -100 ug/mL streptomycin (Life Technologies, NY, USA). The cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. The patient-derived GSCs used in this study were isolated from GBM patients and were well characterized. The patient-derived GSCs were culture in NS-A medium (90% NeuroCult NS-A Basal Medium Human plus 10% Human NeuroCult NS-A proliferation Supplements, StemCell Technologies). Complete medium was supplied with recombinant human epidermal growth factor (R&D system, Minneapolis, MN, USA), and 100 units/mL penicillin plus 100 ug/mL streptomycin (Life Technologies, NY, USA). For differentiation, GSCs were cultured in NS-A medium supplied with 10% fetal bovine serum. Anti-BIRC3 antibody was obtained from R&D system; anti-b-actin IgG-HRP was obtained from Santa Cruz Biotech; and anti-SMAD1, anti-SMAD5, anti-p-SMAD1/5, Goat anti-Rabbit IgG-HRP and Goat anti-mouse IgG-HRP were obtained from CellSignal.

Wu Q., Berglund A.E., MacAulay R.J, & Etame A.B. (2021). A Novel Role of BIRC3 in Stemness Reprogramming of Glioblastoma. International Journal of Molecular Sciences, 23(1), 297.

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GBM Stem-like Cells Culture Protocol

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Mouse 005 GBM stem-like cells (GFP positive) were provided by Dr. I Verma (Salk Institute) and have been described [21 (link), 24 (link)]. They were cultured as spheres in EF20 medium composed of Neurobasal medium (Thermo Fisher Gibco) supplemented with 3 mM l-Glutamine (Corning Mediatech), 1 × B27 supplement (Thermo Flasher Gibco), 0.5 × N2 supplement (Thermo Fisher Gibco), 2 μg/ml heparin (Sigma, St Louis, MO), 20 ng/ml recombinant human epidermal growth factor (R&D Systems, Minneapolis, MN), 20 ng/ml recombinant human fibroblast growth factor-2 (PeproTech, Rocky Hill, NJ), and 0.5 × penicillin G/streptomycin sulfate/amphotericin B complex (Corning Mediatech) at 37 °C and 5% CO₂. To passage cells, neurospheres were dissociated with the Neurocult chemical dissociation kit (Stem Cell Technologies). Mouse GBM GL261 cells were obtained from the National Cancer Institute and grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS). Cells were confirmed to be mycoplasma free (LookOut Mycoplasma kit, Sigma) and used at low passage number.

Li M., Li G., Kiyokawa J., Tirmizi Z., Richardson L.G., Ning J., Das S., Martuza R.L., Stemmer-Rachamimov A., Rabkin S.D, & Wakimoto H. (2020). Characterization and oncolytic virus targeting of FAP-expressing tumor-associated pericytes in glioblastoma. Acta Neuropathologica Communications, 8, 221.

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Mouse Neurofibroma Sphere Culture Protocol

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Mouse neurofibroma/DRG-derived sphere culture was performed as described (26 (link)). Specifically, we plated trypan blue–negative cells at 1 × 10⁴ cells per 24-well low-binding plates in 1 ml of medium containing Dulbecco’s modified Eagle’s medium (DMEM):F-12 (3:1) and recombinant human epidermal growth factor (20 ng/ml; R&D Systems), 20 ng/ml recombinant human basic fibroblast growth factor (R&D Systems), 1% B-27 (Invitrogen), and heparin (2 μg/ml; Sigma-Aldrich). We maintained cultures at 37°C and 5% CO₂. To passage, we centrifuged sphere cultures, dissociated and replated at 1 × 10⁴ cells/ml in fresh sphere medium as described (6 (link)).

Hall A., Choi K., Liu W., Rose J., Zhao C., Yu Y., Na Y., Cai Y., Coover R.A., Lin Y., Dombi E., Kim M., Levanon D., Groner Y., Boscolo E., Pan D., Liu P.P., Lu Q.R., Ratner N., Huang G, & Wu J. (2019). RUNX represses Pmp22 to drive neurofibromagenesis. Science Advances, 5(4), eaau8389.

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Differentiation of SSEA-1+ and SUSD2+ Cells to EEC-like Cells

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P3 SSEA-1⁺ and SUSD2⁺ cells were seeded onto six-well plate at a density of 6 × 10⁵ cells/well in TEM. When reached 90% confluence, cells were cultured in differentiation medium consisting of DMEM/F12 with 5% FBS, 1 × 10^–6 mol/L Estradiol (E2) (Absin, China), 10 ng/ml recombinant human epidermal growth factor (EGF) (R&D Systems, USA), and 10 ng/ml recombinant human platelet-derived growth factor-BB (R&D Systems, USA). The differentiation medium was changed every 2 days for a 20-day differentiation (Fig. 3b). Cytokeratin and Vimentin were tested through immunofluorescence staining. Anti-CD9 and anti-EpCAM were used to detect the results of the EEC-like cells differentiation by flow cytometry. The expressions of pluripotency and epithelium related genes of SSEA-1⁺ and SUSD2⁺ cells before and after differentiation were measured by qPCR analysis.

He W., Zhu X., Xin A., Zhang H., Sun Y., Xu H., Li H., Yang T., Zhou D., Yan H, & Sun X. (2022). Long-term maintenance of human endometrial epithelial stem cells and their therapeutic effects on intrauterine adhesion. Cell & Bioscience, 12, 175.

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Epidermal Growth Factor Signaling Pathway

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Recombinant human epidermal growth factor (EGF) was purchased from R&D Systems. Anti-Grb2 (sc-8034) were purchased from Santa Cruz Biotechnology. Anti-GST (2624), anti-GFP (2956), anti-Tubulin (2146), anti-pEGFR pY1173 (2244), anti-EGFR (4267), anti-Shp2 pY580 (3754), anti-pErk1/2 (4370) and anti-Erk1/2 (9101) were from Cell Signalling Technology. Anti-RFP (A00682) was purchased from Genscript. Anti-6xHis (631210) was purchased from Takara. Anti-Shp2 antibodies were purchased from Santa Cruz Biotechnology, Cell Signal Technology, Sigma or Abcam. Anti-Grb2 pY160 was synthesised from Genscript^{30 (link)}. Metafectin cell transfection reagents were purchased from Biontex.

Lin C.C., Wieteska L., Suen K.M., Kalverda A.P., Ahmed Z, & Ladbury J.E. (2021). Grb2 binding induces phosphorylation-independent activation of Shp2. Communications Biology, 4, 437.

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