Epidermal growth factor egf

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Epidermal growth factor (EGF) is a protein that promotes cell growth and proliferation. It is a key component in cell culture media and is used to support the growth and expansion of various cell types, including stem cells, epithelial cells, and fibroblasts.

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8 protocols using epidermal growth factor egf

Culturing Diverse DMG Cell Lines

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All DMG cell lines used in the study (except for SF7761) were cultured in ultra-low attachment flasks in culture medium with 1:1 ratio of DMEM/F-12 (Invitrogen, 11330-032) and Neurobasal A (Invitrogen, 10888-022) and ten percent each of HEPES Buffer Solution 1 M (Thermo Fisher, 15630080), Sodium Pyruvate solution 100 nM (Life Technologies, 11360070), MEM non-essential amino acids solution 10 mM (Thermo Fisher, 11140050), Glutamax-I Supplement (Thermo Fisher, 35050061), and Penicillin/Streptomycin solution (Life Technologies, 15140122). The media was supplemented with B27 Minus Vitamin A (Invitrogen, 12587-010), epidermal growth factor (EGF; StemCell Tech. Inc., 78006), fibroblast growth factor (FGF; GF003, StemCell Tech., Inc., 78003) and heparin solution, 0.2% (StemCell Tech. Inc., 07980), as well as PDGF-AA (Shenandoah Biotech, 100-16) and PDGF-BB (Shenandoah Biotech, 100-18). SF7761 cell line was cultured in medium with Neurobasal A (Invitrogen, 10888-022) and N-2 Supplement (Invitrogen, 17502), further supplemented with B27 Minus Vitamin A (Invitrogen, 12587-010), epidermal growth factor (EGF; StemCell Tech. Inc., 78006), fibroblast growth factor (FGF; GF003, StemCell Tech. Inc., 78003) and heparin solution, 0.2% (StemCell Tech. Inc., 07980). Cells were dissociated using Accutase (StemCell Tech. Inc., 07922) and passaged every 3 or 4 days.

Khadka P., Reitman Z.J., Lu S., Buchan G., Gionet G., Dubois F., Carvalho D.M., Shih J., Zhang S., Greenwald N.F., Zack T., Shapira O., Pelton K., Hartley R., Bear H., Georgis Y., Jarmale S., Melanson R., Bonanno K., Schoolcraft K., Miller P.G., Condurat A.L., Gonzalez E.M., Qian K., Morin E., Langhnoja J., Lupien L.E., Rendo V., Digiacomo J., Wang D., Zhou K., Kumbhani R., Guerra Garcia M.E., Sinai C.E., Becker S., Schneider R., Vogelzang J., Krug K., Goodale A., Abid T., Kalani Z., Piccioni F., Beroukhim R., Persky N.S., Root D.E., Carcaboso A.M., Ebert B.L., Fuller C., Babur O., Kieran M.W., Jones C., Keshishian H., Ligon K.L., Carr S.A., Phoenix T.N, & Bandopadhayay P. (2022). PPM1D mutations are oncogenic drivers of de novo diffuse midline glioma formation. Nature Communications, 13, 604.

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Mammosphere Formation Assay Protocol

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Mammosphere-forming capacity was performed by plating cells in DMEM/F-12, 0.4% bovine serum albumin (BSA) (VWR), 10 mL/500 mL B27 (Thermofisher) 5 μg/mL insulin, 4 μg/mL heparin (Sagent, Schaumburg, IL), 20 ng/mL fibroblast growth factor 2 (bFGF) (StemCell Technologies), and 20 ng/mL epidermal growth factor (EGF) (StemCell Technologies) into 96-well ultra-low adhesion plates, at a range of cell densities, with EGF, bFGF, and heparin added every 3 days. The number of spheres formed per well was counted and expressed as a percentage of the initial number of cells plated.

Zhang L., Bailleul J., Yazal T., Dong K., Sung D., Dao A., Gosa L., Nathanson D., Bhat K., Duhachek-Muggy S., Alli C., Dratver M.B., Pajonk F, & Vlashi E. (2019). PK-M2-mediated metabolic changes in breast cancer cells induced by ionizing radiation. Breast cancer research and treatment, 178(1), 75-86.

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Quantifying Serum RBP4 Using ELISA

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Non-target shRNA control (SHC002) and RBP4shRNAs (TRCN0000060038 [#1] and TRCN0000060039 [#2]) were purchased from Sigma. STRA6shRNAs (TRCN0000128799 [#2] and TRCN0000129158 [#1]) and EGFP (RHS4459) were from Open Biosystems. Epidermal growth factor (EGF) was from Stem Cell Technologies and basic fibroblast growth factor (FGF) was from Peprotech. Etoposide, insulin, and heparin were purchased from Sigma. Viable cells were counted after trypan blue staining using Countess II FL (Life Technologies). Human RBP4 in serum samples of patients was quantified using an RBP4 Quantikine ELISA kit (R&D Systems) following the manufacturer's protocols.

Karunanithi S., Levi L., DeVecchio J., Karagkounis G., Reizes O., Lathia J.D., Kalady M.F, & Noy N. (2017). RBP4-STRA6 Pathway Drives Cancer Stem Cell Maintenance and Mediates High-Fat Diet-Induced Colon Carcinogenesis. Stem Cell Reports, 9(2), 438-450.

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Culture and Maintenance of Circulating Tumor Cells

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HMCs were purchased from ScienCell Laboratories (Carlsbad, CA, USA) and maintained in poly-L-lysine–coated T175 culture flasks containing complete Meningeal Cell Medium (MenCM) (MenCM + meningeal growth factors + 2% fetal bovine serum (FBS) + penicillin/streptomycin solution [all from ScienCell Laboratories, Carlsbad, CA, USA]). When cells reached 70% confluence, the media was centrifuged at 1500 rpm for 5 minutes, supernatant was collected for generating HMC-conditioned media to culture CSF-CTCs. Cell pellets from CSF was resuspended in a 1:1 ratio of HMC-conditioned media to complete MenCM, with additional 40 ng/ml of fibroblast growth factor (FGF) and 40 ng/ml of epidermal growth factor (EGF) (both Stemcell Technologies, Vancouver, BC, Canada). CSF-CTCs were cultured in single wells of a 96-well plate until confluent, and then transferred to larger culturing apparatus. Culture media was refreshed every 3 days. Melanoma cell line cultures details are in Supplementary Methods.

Law V., Chen Z., Vena F., Smalley I., Macaulay R., Evernden B.R., Tran N., Pina Y., Puskas J., Caceres G., Bayle S., Johnson J., Liu J.K., Etame A., Vogelbaum M., Rodriguez P., Duckett D., Czerniecki B., Chen A., Smalley K.S, & Forsyth P.A. (2022). A preclinical model of patient-derived cerebrospinal fluid circulating tumor cells for experimental therapeutics in leptomeningeal disease from melanoma. Neuro-Oncology, 24(10), 1673-1686.

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Isolation and Culture of Enteric Neuronal Stem Cells

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Enteric neuronal stem/progenitor cells (ENSC) were isolated and cultured as previously described [17 (link), 20 ]. Briefly, gastrointestinal tracts were isolated from postnatal day 14-21 (P14-21) C57BL/6 or DsRed mice. The longitudinal muscle-myenteric plexus (LMMP) was isolated using microdissection. Note that the circular muscle is often included with the LMMP as it adheres to the myenteric plexus. Cells were dissociated with dispase (250 μg ml⁻¹; StemCell Technologies, Vancouver, Canada) and collagenase XI (1 mg ml⁻¹; Sigma Aldrich, St. Louis, MO) at 37°C for 1 hour with gentle pipetting. The cell suspension was passed through a 40 μm cell strainer and cultured at a density of 50,000 cells ml⁻¹ in proliferation medium, consisting of NeuroCult NSC Basal Medium (StemCell Technologies) supplemented with 20 ng ml⁻¹ epidermal growth factor (EGF; StemCell Technologies) and 10 ng ml⁻¹ basic fibroblast growth factor (bFGF; StemCell Technologies) for 7-10 days to form enteric neurospheres. Neurospheres were passaged at day 7 with gentle Accutase (StemCell Technologies) dissociation at 37°C for 20-30 minutes. Cells were re-plated into a 6-well Ultra-low attachment culture plate (Corning, Kennebunk, ME) in conditioned medium (1:2 mix) to obtain the secondary neurospheres that were used in this study.

Hotta R., Cheng L., Graham H.K., Nagy N., Belkind-Gerson J., Mattheolabakis G., Amiji M.M, & Goldstein A.M. (2016). Delivery of enteric neural progenitors with 5-HT4 agonist-loaded nanoparticles and thermosensitive hydrogel enhances cell proliferation and differentiation following transplantation in vivo. Biomaterials, 88, 1-11.

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Glioblastoma Sphere Formation Protocol

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To test for sphere formation, glioblastoma cells were cultured in sphere-inducing media^{35 (link),36 (link)} in analogy to well-used standard protocols.^{70 (link)–74 (link)} Briefly, glioblastoma cells were harvested from T75 flasks and brought to T25 flasks with DMEM/F12 (Gibco/ThermoFisher, Waltham, MA, USA) + 20% BIT (Provitro, Berlin, Germany) +20 ng/ml basic fibroblast growth factor (bFGF) + 20 ng/ml epidermal growth factor (EGF; both Stemcell Technologies, Vancouver, Canada). The two patient-derived lines had been established from trial patients.³⁷ They are available upon reasonable request. NCH421K is a standard gliomasphere cell line^{35 (link),36 (link)} (CSL Cell Lines Service, Eppendorf, Germany). The FAK inhibitor used was 1,2,4,5-benzenetetraamine tetrahydrochloride (Sigma Aldrich, St. Louis, MI, USA).

Erhart F., Hackl M., Hahne H., Buchroithner J., Meng C., Klingenbrunner S., Reitermaier R., Fischhuber K., Skalicky S., Berger W., Spiegl-Kreinecker S., Lötsch D., Ricken G., Kuster B., Wöhrer A., Widhalm G., Hainfellner J., Felzmann T., Dohnal A.M., Marosi C, & Visus C. (2020). Combined proteomics/miRNomics of dendritic cell immunotherapy-treated glioblastoma patients as a screening for survival-associated factors. NPJ Vaccines, 5, 5.

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Sphere Formation from CD133+ Cells

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The sorted CD133⁺ and CD133^- cells were cultured in serum-free medium (NS-A basal medium, Stemcell Technologies, Vancouver, BC, Canada) plus 10% NS-A Proliferation Supplements (Human) (Stemcell Technologies), epidermal growth factor(EGF)(20 ng/mL) (Stemcell Technologies), basic fibroblast growth factor (bFGF)(10 ng/mL) (Stemcell Technologies) and heparin (2 μg/mL) (Invitrogen). The culture medium was replaced every 3 days. The images of spheres were captured after 20 days by microscope and counted numbers under 100×magnification in random visual fields. Some spheres were stained with PE-conjugated CD133/2 (293C3) antibody (Milteny Biotect). Some tumor spheres were dissociated by Collagenase Type Ⅳ(Sigma-Aldrich, St Louis, MO, USA) and then cultured in serum-free medium as mention above to determine whether spheres could form again.

Zhi Y., Mou Z., Chen J., He Y., Dong H., Fu X, & Wu Y. (2015). B7H1 Expression and Epithelial-To-Mesenchymal Transition Phenotypes on Colorectal Cancer Stem-Like Cells. PLoS ONE, 10(8), e0135528.

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Isolation and Culture of Embryonic and Neonatal Neural Progenitor Cells

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Embryonic NPCs were isolated from pregnant NOD/SCID mice (n = 3) on E13.5 day of gestation. Striata were dissected from each embryo in phosphate-buffered saline (PBS) containing 0.6% glucose (Sigma-Aldrich) and penicillin-streptomycin (50 U/ml; Life Technologies). After dissection, the tissue was triturated to a single cell suspension using a transfer pipette. Cells were cultured in NeuroCult™ Proliferation Medium containing 20 ng/ml epidermal growth factor (EGF) (Stemcell Technologies Inc., Vancouver, BC, Canada). NPCs from infant NOD/SCID mice were isolated from the ipsilateral and contralateral SVZ at 1 week post-injury (n = 5). The SVZ tissue was triturated with neural tissue dissociation solution (Sumitomo Dainippon Pharma Co., Ltd, Tokyo, Japan). Cells were cultured in NeuroCult™ Proliferation Medium containing 20 ng/ml EGF, 10 ng/ml fibroblast growth factor (FGF) and 2 μl/ml heparin (Stemcell Technologies Inc.). Cells were proliferated to generate neurospheres.

Wang F., Baba N., Shen Y., Yamashita T., Tsuru E., Tsuda M., Maeda N, & Sagara Y. (2017). CCL11 promotes migration and proliferation of mouse neural progenitor cells. Stem Cell Research & Therapy, 8, 26.

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