Epidermal growth factor (egf)

Manufactured by American Type Culture Collection

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Epidermal Growth Factor (EGF) is a protein that stimulates cell growth and differentiation. It is a key regulator of cell proliferation, survival, and migration. EGF binds to its receptor (EGFR) on the cell surface, triggering a signaling cascade that promotes cellular processes.

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9 protocols using epidermal growth factor (egf)

Culturing HK-2 Cells in DMEM/F-12

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HK‐2 cells (CRL‐2190, ATCC) were cultured in DMEM/F‐12 media containing 10% heat‐inactivated fetal bovine serum, bovine pituitary extract (0.05 mg/ml), epidermal growth factor (5 ng/ml), and antibiotic‐antimycotic. All cell culture reagents were purchased from Thermo Fisher Scientific.

Patel S, & Hussain T. (2020). Synergism between Angiotensin receptors ligands: Role of Angiotensin‐(1‐7) in modulating AT2R agonist response on nitric oxide in kidney cells. Pharmacology Research & Perspectives, 8(6), e00667.

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Cell Culture Protocols for Diverse Cell Lines

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All cell culture media were supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin unless otherwise stated. Jurkat cells (JE6-1) were cultured in RPMI 1640 medium (GlutaMAX; Gibco). Caco-2 (ATCC) cells – epithelial cells derived from a colon adenocarcinoma – were cultured in Dulbecco's modified Eagle's medium (DMEM, GlutaMAX, high-glucose; Gibco) supplemented with 1% non-essential amino acids and FHs 74 Int cells (ATCC) – epithelial cells derived from healthy fetal small intestine – were cultured in Hybri-Care medium (ATCC) supplemented with 30 ng/mL epidermal growth factor (Biozym). BeWo cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GlutaMAX, high-glucose; Gibco). All cell lines were cultivated at 37 °C in a humidified atmosphere (95%) containing 5% CO₂. Cultured cells were washed with PBS and lysed in cell lysis buffer (150 mM NaCl, 50 mM Tris pH8, 1% Triton X-100) containing protease inhibitor (Roche). Protein concentrations were determined by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).

Schwertner K., Gelles K., Leitner J., Steinberger P., Gundacker C., Vrticka R., Hoffmann-Sommergruber K., Ellinger I, & Geiselhart S. (2023). Human intestine and placenta exhibit tissue-specific expression of RAGE isoforms. Heliyon, 9(8), e18247.

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Culture and Maintenance of Intestinal Cell Lines

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Nontransformed human small intestinal epithelial FHs 74 Int cells (ATCC, Manassas, VA, USA) were maintained in Hybri‐Care medium (ATCC, Manassas, VA, USA) supplemented with 10% heat‐inactivated fetal bovine serum (Sigma–Aldrich, Zwijndrecht, The Netherlands), 50 µg mL⁻¹ penicillin‐streptomycin solution (Sigma‐Aldrich, Zwijndrecht, The Netherlands), and 30 ng mL⁻¹ EGF (ATCC, Manassas, VA, USA). Human colon carcinoma T84 cells were cultured in Dulbecco's Modified Eagle Medium:F‐12 (DMEM:F12) medium (Gibco, Life Technologies, Bleiswijk, The Netherlands) supplemented with 10% heat‐inactivated fetal bovine serum (Sigma‐Aldrich, Zwijndrecht, The Netherlands) and 50 mg mL⁻¹ gentamicin (Lonza, Verviers, Belgium). Cells were cultured at 37 °C in 5% CO₂ as recommended by the manufacturer. Recombinant human TNF‐α was obtained from PeproTech (Rocky Hill, NJ, USA).

Cheng L., Kong C., Wang W., Groeneveld A., Nauta A., Groves M.R., Kiewiet M.B, & de Vos P. (2021). The Human Milk Oligosaccharides 3‐FL, Lacto‐N‐Neotetraose, and LDFT Attenuate Tumor Necrosis Factor‐α Induced Inflammation in Fetal Intestinal Epithelial Cells In Vitro through Shedding or Interacting with Tumor Necrosis Factor Receptor 1. Molecular Nutrition & Food Research, 65(7), 2000425.

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Established Glioma Cell Line Characterization

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MB002 and MB004 were gifts from Y.J. Cho (Oregon Health and Science University, Portland, OR, United States). D556^{58 (link)} was a gift from D. Bigner (Duke School of Medicine). D556 and D283 (D. Bigner, ATCC) cell lines were maintained in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin and streptomycin (ThermoFisher Scientific). MB002 and MB004 cells were maintained in culture medium with 1:1 DMEM/F12 (Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12) and Neurobasal™-A Medium supplemented with non-essential amino acids, Sodium Pyruvate, HEPES, GlutaMax, B27, EGF, FGF, Heparin, LIF, 10% fetal bovine serum (ATCC), and 100 U/mL penicillin and streptomycin (All from ThermoFisher Scientific). All cell lines were maintained at 37 °C with 5% CO₂ in a 95% humidified atmosphere. All established cell lines were verified with STR analysis (GRCF, Johns Hopkins). Antibodies used in this study are listed in Supplementary Data 10.

Rivero-Hinojosa S., Grant M., Panigrahi A., Zhang H., Caisova V., Bollard C.M, & Rood B.R. (2021). Proteogenomic discovery of neoantigens facilitates personalized multi-antigen targeted T cell immunotherapy for brain tumors. Nature Communications, 12, 6689.

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Culturing Breast Cancer Cell Lines

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Human breast cancer cell lines T-47D (HTB-113; ATCC), Hs 578T (HTB-126; ATCC) and MDA-MB-231 (HTB-26; ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. MCF-10-2A (CRL-10781; ATCC) cells were grown in DMEM-F12 medium supplemented with 5% horse serum, 1% penicillin/streptomycin, 20 ng/ml epidermal growth factor (EGF), 100 ng/ml cholera toxin, 0.01 mg/ml insulin and 500 ng/ml hydrocortisone. All cell lines were maintained at 37°C in a humidified atmosphere with 5% CO₂. For all experiments, cells were seeded in 6-well at a concentration of 1.5×10⁵ cells/ml for 24 h experiments and 1×10⁵ cells/ml for 48 h experiments, with the exception of the ECAR experiments in which cells were seeded in XF 24-well plates at a concentration of 1×10⁴ cells/well. All medium constituents were acquired from Biochrom with the exception of EGF, cholera toxin, hydrocortisone and insulin that were purchased from Sigma-Aldrich.

Pereira C.S., Guedes J.P., Gonçalves M., Loureiro L., Castro L., Gerós H., Rodrigues L.R, & Côrte-Real M. (2016). Lactoferrin selectively triggers apoptosis in highly metastatic breast cancer cells through inhibition of plasmalemmal V-H+-ATPase. Oncotarget, 7(38), 62144-62158.

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Organoid Culture Medium Preparation

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Cell lines for producing conditioned media were tested for the presence of mycoplasma (Lonza MycoAlert PLUS kit, LT07–701). Media containing Wnt3A, EGF, Noggin and R-spondin1 (WENR media) was generated by harvesting conditioned media from cultured L-WRN cells (ATCC CRL-3276) at 2X dilution in DMEM and supplemented with 50 ng/mL recombinant mouse EGF (ThermoFisher; PMG8041) as previously described (Miyoshi and Stappenbeck, 2013 (link)). Media containing EGF, Noggin and R-spondin1 but not Wnt3A (ENR media) was prepared by first harvesting conditioned media from cultured 293T-HA-RspoI-Fc cells (Trevigen Cat# 3710–001-01) as described (R&D Systems Protocol; https://resources.rndsystems.com/images/site/dw_r-spondinmediumprotocol_34749-web.pdf?v=1). 10 mL of conditioned media was then added to 86 mL DMEM/F12 (ThermoFisher, Cat# 11320–033), 1 mL Glutamax (ThermoFisher, Cat# 35050–079), 1 mL HEPES (ThermoFisher, Cat# 15630–080), 200 μL Primocin (Invivogen, ant-pm-1), 1 mL B27 (Thermo, Cat# 17504–044), 0.5 mL N2 (ThermoFisher, Cat# 17502–048), 250 μl N-acetyl cysteine 500 mM stock solution (Sigma, SKU-A7250–10G), with EGF at final concentration (50 ng/mL), and Noggin (MilliporeSigma; SKU-SRP3227–20UG) at final concentration 100 ng/mL. Media were filter-sterilized and stored for up to 6 months at −20°C. 50 mL aliquots of media were thawed and stored at 4°C as necessary to feed organoids.

Tallapragada N.P., Cambra H.M., Wald T., Jalbert S.K., Abraham D.M., Klein O.D, & Klein A.M. (2021). Inflation-collapse dynamics drive patterning and morphogenesis in intestinal organoids. Cell stem cell, 28(9), 1516-1532.e14.

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Maintenance of Human Breast Cell Lines

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Nontumorigenic human breast epithelial cell line MCF10A was purchased from ATCC (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 5% horse serum, 20 ng·mL⁻¹ EGF, 10 μg·mL⁻¹ insulin, and 500 ng·mL⁻¹ hydrocortisone. DMEM/F12 was purchased from Thermo Fisher Scientific (Waltham, MA, USA), and EGF, insulin, and hydrocortisone were purchased from Sigma (St. Louis, MO, USA). Human breast carcinoma MDA‐MB‐453 cells and SKBR3 cells were kindly provided by Robert I. Glazer in Georgetown University. Human embryonic kidney cell line 293T was kindly provided by Uda in Niigata University of Pharmacology and Applied Life Sciences. MDA‐MB‐453, SKBR3, and 293T cells were maintained in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific).

Hasegawa T., Adachi R., Iwakata H., Takeno T., Sato K, & Sakamaki T. (2017). ErbB2 signaling epigenetically suppresses microRNA‐205 transcription via Ras/Raf/MEK/ERK pathway in breast cancer. FEBS Open Bio, 7(8), 1154-1165.

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Endothelial Cell Culture and Chemotaxis Assay

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Primary human aortic endothelial cells (HAECs) and vascular cell basal medium supplemented with V EGF (5 ng/mL), EGF (5 ng/mL), FGF (5 ng/mL), IGF-1 (15 ng/mL), L-glutamine (10 mM), heparin sulfate (0.75 U/mL), hydrocortisone (1 µg/mL), ascorbic acid (50 µg/mL), fetal bovine serum (FBS), penicillin (10,000 U/mL), streptomycin (10 mg/mL) and amphotericin B (25 µg/mL) were all obtained from American Type Culture Collection (ATCC, Manassas, VA). Acetylated PGP and PGG were purchased from Bachem (Torrance, CA) and purified to neutrophil chemotactic activity. Human IL-8 with carrier was obtained from Cell Signaling Technology (Danvers, MA). Finally, the selective CXCR2 antagonist SB225002 was purchased from Sigma Aldrich (St. Louis, MO).

Payne G.A., Li J., Xu X., Jackson P., Qin H., Pollock D.M., Wells J.M., Oparil S., Leesar M., Patel R.P., Blalock J.E, & Gaggar A. (2017). The Matrikine Acetylated Proline-Glycine-Proline Couples Vascular Inflammation and Acute Cardiac Rejection. Scientific Reports, 7, 7563.

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Culturing Breast Cancer Cell Lines

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MCF12A cells (American Type Culture Collection) were grown in DMEM/F12 supplemented with 10 mM HEPES, 10 μg/ml insulin, 20 ng/ml EGF, 20 ng/ml cholera toxin, 30, mM sodium bicarbonate, 0.5 μg/ml hydrocortisone and 5% normal horse serum in a humidified atmosphere containing 5% CO₂ at 37 °C. MCF7 cells (ATCC) were grown in Improved Minimum Essential Medium (IMEM) (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum. HRG was purchased from R&D Systems (Minneapolis, MN, USA). EGF was purchased from Peprotech (Rocky Hill, NJ, USA). EHT1864 was purchased from Tocris (Minneapolis, MN, USA).

Goka E.T, & Lippman M.E. (2015). Loss of the E3 ubiquitin ligase HACE1 results in enhanced Rac1 signaling contributing to breast cancer progression. Oncogene, 34(42), 5395-5405.

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