Human epidermal growth factor (hegf)

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The HEGF is a laboratory equipment product by Thermo Fisher Scientific. It serves as a core function to perform specific laboratory tasks. No further details can be provided in an unbiased and factual manner.

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194 protocols using human epidermal growth factor (hegf)

Functionalized DPP Scaffold for hEGF Delivery

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The DPP was incubated in EDC/NHS (Thermo Scientific, Massachusetts, USA) solution for 20 min after full rehydration, followed with immersion in a 60 mg/ml saturated Asp solution by ultrasound with 37 Hz for 4 h and then sequentially incubated for 20 h under shaking at RT. Then, the DPP-Asp was immersed in 200 ng/ml hEGF (Thermo Scientific) solution at 4°C overnight, and DPP-Asp-hEGF was prepared. These scaffolds are stored at −20°C until use.

Chen F., Deng J., Luo L., Zhu Y., Dong Y., Yang Y., Zhang R., Chen J, & Zhou Q. (2022). Crosslinked Decellularized Porcine Pericardium as a Substrate for Conjunctival Reconstruction. Stem Cells International, 2022, 7571146.

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Cell Culture Reagents and Compounds

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DMEM (high glucose) and horse serum were from Sigma‐Aldrich; FBS was from Gibco; l‐glutamine, penicillin, trypsin and streptomycin were from Euroclone; matrigel was from Corning; and gentamycin, fetuin, hEGF, bFGF and trypsin were from Life Technologies. Insulin and dexamethasone, Yoda1, gadolinium and Fura‐2 AM were from Sigma‐Aldrich; Medium 199, gentamycin, fetuin, hEGF and bFGF were from Life Technologies; and Alexa‐488‐α‐bungarotoxin was from Invitrogen.

Bosutti A., Giniatullin A., Odnoshivkina Y., Giudice L., Malm T., Sciancalepore M., Giniatullin R., D'Andrea P., Lorenzon P, & Bernareggi A. (2021). “Time window” effect of Yoda1‐evoked Piezo1 channel activity during mouse skeletal muscle differentiation. Acta Physiologica (Oxford, England), 233(4), e13702.

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Evaluating Ovarian Cancer Stem Cell Frequency

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OV90, OVCAR3 and OVCAR4 cells were plated in triplicate (3.5x10⁵ cells/well) in 6 well plates and treated after 24 hours with either 10 nM S3F-CL-MMAE or the vehicle control. After 3 or 6 days, cells were harvested, re-suspended in in serum-free DMEM/F-12, HEPES medium with 1X B-27 supplement (Gibco), 10 ng/mL human epidermal growth factor (Life Technologies) and 20 ng/mL human β fibroblast growth factor (Life Technologies) and plated in low adhesion 96 well plates at concentrations of 1, 10, 20 and 40 cells/well. After 7-8 days in culture, the wells were evaluated for presence or absence of tumorspheres. Tumor cell aggregates were not counted as positive unless individual tumorspheres were identified. Stem cell frequencies were then calculated as described (http://bioinf.wehi.edu.au/software/elda/).

Starbuck K., Al-Alem L., Eavarone D.A., Hernandez S.F., Bellio C., Prendergast J.M., Stein J., Dransfield D.T., Zarrella B., Growdon W.B., Behrens J., Foster R, & Rueda B.R. (2018). Treatment of ovarian cancer by targeting the tumor stem cell-associated carbohydrate antigen, Sialyl-Thomsen-nouveau. Oncotarget, 9(33), 23289-23305.

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Establishment and Characterization of Pancreatic Cancer Cell Lines

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Human cell lines PANC-1 and HPDE were obtained from American Type Culture Collection (ATCC, Rockville, MD) or as a generous gift from Mokenge Malafa (Moffitt Cancer Center, Tampa, FL), respectively. L3.6pl metastatic variant was derived as previously described [17 (link), 18 (link)]. The selection of L3.6pl^GemRes gemcitabine-resistant pancreatic cancer cells was conducted as previously described [19 (link)]. All human PC cell lines were authenticated within 6 months by short tandem repeat (STR) analysis. All cells were maintained and stimulated in high-nutrition media [Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) Advanced (Life Technologies, Carlsbad, CA), 10 % fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA), 4 mM GlutaMAX^™ (Life Technologies), 20 ng/mL human epidermal growth factor (Life Technologies), 40 ng/mL dexamethasone (Sigma-Aldrich, St. Louis, MO) and antibiotic antimycotic solution (Sigma)] in 5 % CO₂/95 % air at 37 °C. The creation of L3.6pl^CXCL10 CXCL10-constitutively expressing cells were created by transfection with pCMV6.puromycin vector (Origene, Rockville, MD) containing human CXCL10 cDNA and grown in selective medium containing puromycin (10 μg/mL). Isolated clones were expanded, and CXCL10 secretion was confirmed by ELISA (BD Biosciences, Franklin Lakes, NJ).

Delitto D., Perez C., Han S., Gonzalo D.H., Pham K., Knowlton A.E., Graves C.L., Behrns K.E., Moldawer L.L., Thomas R.M., Liu C., George TJ J.r., Trevino J.G., Wallet S.M, & Hughes S.J. (2015). Downstream mediators of the intratumoral interferon response suppress antitumor immunity, induce gemcitabine resistance and associate with poor survival in human pancreatic cancer. Cancer immunology, immunotherapy : CII, 64(12), 1553-1563.

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Pancreatic Tumor Cell Culture Protocol

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Tumor cells were cultured from a primary pancreatic tumor isolated from a 17 week old RT2 AB6F1 male mouse; this mouse showed liver metastasis at the time of euthanasia. To deter growth of normal fibroblasts, the tumor cells were grown in a stem cell medium for several weeks, consisting of Dulbecco's modified Eagle's medium/F12 medium (Life Technologies) supplemented with N-2 supplement (Life Technologies), 20 ng/ml human epidermal growth factor (Life Technologies), 10 ng/ml human basic fibroblast growth factor (Sigma-Aldrich), 4 μg/ml heparin (Sigma-Aldrich), 4 mg/ml bovine serum albumin (Life Technologies), 20 μg/ml human insulin, zinc solution (Life Technologies), and 2.9 mg/ml glucose (Sigma-Aldrich) [46 (link)]. After several passages, the cells were switched to RPMI media with 10% fetal bovine serum. SV40 T-antigen antibody (Abcam), insulin antibody (Cell signaling) and CD88 antibody (Biolegend) were used for flow cytometry analysis. Invasiveness was tested using a matrigel invasion chamber (Corning) with or without addition of recombinant mouse C5a protein (R&D Systems). The invasion assay was repeated three times.

Contractor T., Kobayashi S., da Silva E., Clausen R., Chan C., Vosburgh E., Tang L.H., Levine A.J, & Harris C.R. (2016). Sexual dimorphism of liver metastasis by murine pancreatic neuroendocrine tumors is affected by expression of complement C5. Oncotarget, 7(21), 30585-30596.

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Culturing TNBC and Normal Breast Cells

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Normal human breast epithelial cells (MCF-10A) and two TNBC cell lines (MDA-MB231 and MDA-MB468) were used in this work. MDA-MB231 and MDA-MB468 cells were grown in RPMI 1640 (Lonza) supplemented with penicillin/streptomycin (100×) (Euroclone), fetal bovine serum (10%) (Invitrogen), and nonessential amino acids (100×) (Invitrogen), and Glutamax (100×) (Invitrogen) whereas MCF-10A cells in DMEM were supplemented with 20 ng/mL human epidermal growth factor (Life Technologies), 10 μg/mL human insulin (Life Technologies Corporation), and 0.5 μg/mL of hydrocortisone (Sigma-Aldrich). In the case of MDA-MB468 cells, Ham’s F-12 medium was also added to the medium (1:1 ratio). Finally, the cells were kept at 37 °C in an incubator in a humidified atmosphere (5% CO₂ and 95% air).

Nunziata C., Polo A., Sorice A., Capone F., Accardo M., Guerriero E., Marino F.Z., Orditura M., Budillon A, & Costantini S. (2019). Structural analysis of human SEPHS2 protein, a selenocysteine machinery component, over-expressed in triple negative breast cancer. Scientific Reports, 9, 16131.

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Isolation and Characterization of Patient-Derived Cell Lines

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Patient-derived PC cell lines were isolated from patient-derived xenografts as previously described (16 (link),17 (link)). Epithelial cell purity was confirmed by expression of HLA ABC, EpCam and cytokeratins 18 and 19 (Biolegend, San Diego, CA). Patient-derived tumor associated stroma (TAS) cell lines were generated as previously described (15 (link)). Cell purity was confirmed by uniform expression of α-smooth muscle actin (R&D Systems, Minneapolis, MN) by flow cytometry and immunocytochemistry. Both PC and TAS cell lines were maintained and stimulated in growth medium [Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12) Advanced (Life Technologies, Carlsbad, CA), 10% fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA), 4 mM GlutaMAX™ (Life Technologies), 20 ng/mL human epidermal growth factor (Life Technologies), Primocin™ (Invivogen, San Diego, CA) and antibiotic antimycotic solution (Sigma)] in 5% CO2/95% air at 37°C.

Delitto D., Delitto A.E., DiVita B.B., Pham K., Han S., Hartlage E.R., Newby B.N., Gerber M.H., Behrns K.E., Moldawer L.L., Thomas R.M., George TJ J.r., Brusko T.M., Mathews C.E., Liu C., Trevino J.G., Hughes S.J, & Wallet S.M. (2016). Human Pancreatic Cancer Cells Induce a MyD88-Dependent Stromal Response To Promote a Tumor-Tolerant Immune Microenvironment. Cancer research, 77(3), 672-683.

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Sphere Formation and Phenotypic Analysis

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OV90, OVCAR3, and OVCAR4 cells were plated in either their respective normal cell culture conditions or in ultralow attachment six-well plates (Sigma-Aldrich) at a density of 2 × 10⁴ cells/well in serum-free DMEM/F-12, HEPES medium with 1X B-27 supplement (Gibco), 10 ng/mL human epidermal growth factor (Life Technologies) and 20 ng/mL human β fibroblast growth factor (Life Technologies). Spheres were harvested after 10-12 days. Media was discarded and spheres were re-suspended in 0.25% trypsin. Cells plated under regular culture conditions as well as the sphere promoting conditions were stained for STn and CD133 and analyzed by flow cytometry as described.

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Breast Cancer Cell Line Cultivation

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Three human breast cancer cell lines, MDA-MB231, MDA-MB468 and MCF-7, all derived from adenocarcinoma metastasis and on normal human breast epithelial cells MCF-10A were used. In particular, MCF-7 and MCF-10A cells were expanded at 37 °C in a humidified atmosphere of 5% CO₂ in culture medium DMEM (Dulbecco’s Modified Eagle’s Medium, Lonza), whereas MDA-MB-231 and MDA-MB-468 in RPMI 1640 (Lonza, Munchensteinerstrasse, Basel, Switzerland), supplemented with fetal bovine serum (FBS) (Invitrogen, Camarillo, CA, USA) at 10%, Penicillin/Streptomycin 100x (Euroclone, Devon, UK), Glutamax 100x (Invitrogen) non-essential amino acids 100x (Invitrogen). Moreover, in the case of MCF-10A the DMEM was supplemented also with human insulin 10 μg/mL (Life Technologies Corporation, Carlsbad, CA, USA), human epidermal growth factor 20 ng/mL (Life Technologies), and hydrocortisone 0.5 μg/mL (Sigma-Aldrich, St. Louis, MO, USA) according to the procedure reported in Rothwell et al. (2014), while for MDA-MB468 the medium was implanted with Ham’s F-12 medium (1:1 mixture). Phosphate buffer (PBS Phosphate buffered saline Ca²⁺ and Mg²⁺ free) and trypsin (Ca²⁺ and Mg²⁺ free) were supplied by Euroclone. Finally, the cells were kept in an incubator at a humidified atmosphere of 95% air and 5% CO₂ at 37 °C.

Costantini S., Guerriero E., Teta R., Capone F., Caso A., Sorice A., Romano G., Ianora A., Ruocco N., Budillon A., Costantino V, & Costantini M. (2017). Evaluating the Effects of an Organic Extract from the Mediterranean Sponge Geodia cydonium on Human Breast Cancer Cell Lines. International Journal of Molecular Sciences, 18(10), 2112.

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Cell Culture Protocol for HeLa, HUVEC, and HMEC-1

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HeLa and HUVEC cells were grown as described previously [4 (link)]. HMEC-1 cells were grown in MCDB-131 medium (Life Technologies, Germany) supplemented with 10% fetal bovine serum (Life Technologies), 1% Glutamax (Life Technologies), 10 ng·mL⁻¹ human epidermal growth factor (Life Technologies) and 1 µg·mL⁻¹ hydrocortisone (Sigma-Aldrich). Cells were kept in a humidified 5% CO₂ atmosphere at 37 °C.

Torrano A.A, & Bräuchle C. (2014). Precise quantification of silica and ceria nanoparticle uptake revealed by 3D fluorescence microscopy. Beilstein Journal of Nanotechnology, 5, 1616-1624.

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