Mouse egf

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Mouse EGF is a recombinant protein that functions as a growth factor. It stimulates the proliferation of a variety of cell types, including epithelial, endothelial, and fibroblast cells.

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Recombinant mouse EGF (20 citations)

27 protocols using mouse egf

Culturing Endocervical and Ectocervical Cells

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Endocervical cells were cultured in ADF medium (Invitrogen, 12634) supplemented with 12 mM HEPES, 1% GlutaMax, 1% B27, 1% N2, 50 ng ml⁻¹ mouse EGF (Invitrogen, 15630-056, 35050-038, 17504-044, 17502048, PMG8043), 100 ng ml⁻¹ mouse noggin, 100 ng ml⁻¹ human FGF10 (Peprotech, 250-38-100, 100-26-25), 1.25 mM N-acetyl-l-cysteine, 10 mM nicotinamide, 10 µM ROCK inhibitor (Y-27632) (Sigma, A9165-5G, N0636, Y0503), 2 µM TGF-β receptor kinase inhibitor IV (Calbiochem, 616454), 1% penicillin–streptomycin (Gibco, 15140-122) with 25% WNT3A- and 25% R-spondin-1-conditioned medium. Ectocervical cells were cultured in endocervical medium but without 25% WNT3A- and 25% R-spondin 1-conditioned medium.

Chumduri C., Gurumurthy R.K., Berger H., Dietrich O., Kumar N., Koster S., Brinkmann V., Hoffmann K., Drabkina M., Arampatzi P., Son D., Klemm U., Mollenkopf H.J., Herbst H., Mangler M., Vogel J., Saliba A.E, & Meyer T.F. (2021). Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia. Nature Cell Biology, 23(2), 184-197.

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SCAMP3 Regulation of EGF Signaling

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Mouse EGF and rapamycin were purchased from Invitrogen (Carlsbad, CA, USA) and used at a working concentration of 100ng/mL and 20nM, respectively. Gefitinib was purchased from VWR (Philadelphia, PA, USA) and used at a working concentration of 1μM. The pcDNA3.0-SCAMP3 plasmid construct was generated as described by others.11 (link) The sequence of SCAMP3-siRNA oligonucleotide was 5′-CCUAAGAACUAUGGCUCAUTT-3′.12 (link) The antibodies used in this study include anti-SCAMP3 (Proteintech, 26888-1-AP), anti-Ki67 (Proteintech, 27309-1-AP), anti-EGFR, anti-p70-S6K (Cell Signaling, cat# 2708), anti-phospho-p70-S6K (Cell Signaling, cat# 9234), anti-4E-BP1 (Cell Signaling, cat# 9644), anti-phospho-4E-BP1 (Cell Signaling, cat# 2855), anti-GAPDH (Cell Signaling, cat# 5174).

Li C., Zhang Z., Lv P., Zhan Y, & Zhong Q. (2020). SCAMP3 Promotes Glioma Proliferation and Indicates Unfavorable Prognosis via Multiple Pathways. OncoTargets and therapy, 13, 3677-3687.

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Establishment and Infection of Neurosphere Cultures

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To prepare a neurosphere culture, the subventricular zone was dissected from neonatal mice and enzymatically dissociated with 0.25% trypsin and bovine DNase-I. Cells were passed through a 70 µm cell strainer followed by a 40 µm cell strainer and then plated with proliferation media consisting of neurosphere basal media (low-glucose DMEM, F12, HEPES, penicillin/streptomycin) supplemented with B27, mouse EGF, and human FGF2 (Invitrogen). To induce differentiation of neurospheres, growth factors were removed and N2 was added to the neurosphere basal media along with B27. Neurospheres were re-fed every 48 hr and used for up to 12 passages.
To infect neurospheres with LCMV, the cells were first plated in 24-well plates at a density of 125,000 cells/well on poly-d-lysine/laminin-coated coverslips (Invitrogen). The virus was diluted in neurosphere basal media and used to infect cells at 1 M.O.I. Infected cell cultures were incubated in a BSL-II primary tissue culture incubator at 37°C with 5% CO₂. To perform quantitative immunocytochemistry, cell cultures were fixed for 15 min at room temperature in 4% PFA and incubated in primary antibodies for 1 hr at room temperature. Secondary antibodies were then added for 1 hr at room temperature.

Sun T., Vasek M.J, & Klein R.S. (2014). Congenitally Acquired Persistent Lymphocytic Choriomeningitis Viral Infection Reduces Neuronal Progenitor Pools in the Adult Hippocampus and Subventricular Zone. PLoS ONE, 9(5), e96442.

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Stem Cell Culture Protocols

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ESCs were grown on gelatin-coated plates in 2i media: 1:1 neurobasal and Dulbecco’s Modified Eagle’s Medium (DMEM)/F12, supplemented with 0.5× N2 (Thermo Scientific, #17502048), 0.5× B27 (Thermo Scientific, #17504044), 0.05% BSA (Thermo Scientific, #15260037), 1 mM PD0325901 (Stemcell Technologies, #72182), 3 μM CHIR99021 (Miltenyi Biotec, #130-103-926), 2 mM L-glutamine, 0.15 mM monothioglycerol, 100 U/ml LIF. NSCs were grown on laminin-coated (Sigma Aldrich, #L2020) plates in DMEM/F12 medium supplemented with 2 mM L-glutamine, 0.5× N2, B27, glucose, BSA, HEPES, and 10 ng/ml of both mouse EGF (Peprotech, #315-09) and human FGF-2 (Peprotech, #100-18B). MEFs were cultured in DMEM supplemented with 10% fetal bovine serum (FBS).

Haward F., Maslon M.M., Yeyati P.L., Bellora N., Hansen J.N., Aitken S., Lawson J., von Kriegsheim A., Wachten D., Mill P., Adams I.R, & Caceres J.F. (2021). Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function. eLife, 10, e65104.

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Isolation and Culture of Optic Glioma Cells

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Optic glioma cells were isolated from the optic nerves of three-month-old Nf1^OPG mice^{18 (link),32 (link)}. Single cells were dissociated from the OPG with a digestion medium containing 0.01% sodium bicarbonate, 15 mM HEPES, 0.5% glucose, 0.2% BSA, 0.004% DNase I and 0.01% trypsin in HBSS. The reaction was quenched using 10% fetal bovine serum, followed by two washes in HBSS. The cell pellets were suspended and grown in low-attachment dishes (Millipore Sigma CLS3262) supplemented with neural stem cell (NSC) medium containing 61% DMEM low glucose medium, 35% neurobasal-A (Invitrogen 10888-022), 2 mM GlutaMAX (Invitrogen 35050-061), 1% penicillin–streptomycin, 1% N2 (Thermo Fisher Scientific 17502001), 2% B27-A (Thermo Fisher Scientific 12587-010), 20 ng ml⁻¹ mouse FGF (PeproTech 450-33), and 20 ng ml⁻¹ mouse EGF (PeproTech 315-09). Cells were authenticated by staining for OPG markers (SOX2, nestin and CD133) and neurosphere-forming assays^{18 (link),32 (link)}. Glioma neurospheres were passaged using Accutase (STEMCELL Technologies 07920, 15-min incubation at room temperature) to achieve single-cell suspension. Mycoplasma testing was performed periodically. For the Nlgn3 qRT–PCR experiment, optic glioma cells were incubated with 30 nM NLGN3 in the NSC medium supplemented with 2% B27-A for 24 h.

Pan Y., Hysinger J.D., Barron T., Schindler N.F., Cobb O., Guo X., Yalçın B., Anastasaki C., Mulinyawe S.B., Ponnuswami A., Scheaffer S., Ma Y., Chang K.C., Xia X., Toonen J.A., Lennon J.J., Gibson E.M., Huguenard J.R., Liau L.M., Goldberg J.L., Monje M, & Gutmann D.H. (2021). NF1 mutation drives neuronal activity-dependent initiation of optic glioma. Nature, 594(7862), 277-282.

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Organoid Infection and Analysis with WNT3A and Nicotinamide

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Freshly split CKIa/p53 DKO and Apc^Min/Min organoids were treated with 80 ng WNT3A (Peprotech) and 10 mM nicotinamide (Sigma-Aldrich) for three days before infection. On the day of infection, organoids were incubated in 200 μl of concentrated virus in infection medium (DMEM/F12 supplemented with WNT3A (80 ng/ml, Peprotech), 10 mM nicotinamide, B27 (1:50, Gibco), mouse Noggin (100 ng/ml, Peprotech), mouse EGF (20 ng/ml, Peprotech), human basic FGF (10 ng/ml, Peprotech) human R-spondin-1 (500 ng/ml, Peprotech), ROCK inhibitor (Sigma-Aldrich) and polybrene (8 μg /ml, Sigma-Aldrich) for 4 h. Infected organoids were collected, Matrigel-embedded and grown in DMEM/F12 medium for five days. Immunofluorescent double staining for p53–GFP and Ki67–GFP was performed and the percentage of organoids with outpocket budding was calculated out of the total GFP-expressing population of organoids. Two independent orga-noid infection cultures were analysed in Fig. 3b. Bright-field and GFP imaging were performed using a fluorescent microscope (Observer Z1, Zeiss).

Kadosh E., Snir-Alkalay I., Venkatachalam A., May S., Lasry A., Elyada E., Zinger A., Shaham M., Vaalani G., Mernberger M., Stiewe T., Pikarsky E., Oren M, & Ben-Neriah Y. (2020). The gut microbiome switches mutant p53 from tumour-suppressive to oncogenic. Nature, 586(7827), 133-138.

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Canine Bladder Cancer Organoid Culture

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In the current investigation, DBCO was produced using aseptically obtained urine samples from four dogs with BC illness. The samples were transferred immediately in a cooled shipping medium to our laboratory. Four strains of DBCO were generated as mentioned earlier in our previous articles (Elbadawy et al., 2019 (link); Elbadawy et al., 2021b (link); Elbadawy et al., 2022 (link)). The details of culture conditions, medium, supplements, growth factors, processing, and passages of the DBCO were the same. Concisely, the culture medium was Advanced DMEM including 50% Wnt, R-spondin, and Noggin conditioned medium; 100 μg/mL Primocin; 10 mM nicotinamide; 1% GlutaMax; and 1 mM N-Acetyl-L-cysteine (Thermo Fisher Scientific, Waltham, MA, United States); 500 nM A83-01 (Adooq Bioscience, Irvine, CA, United States); and 50 ng/mL mouse EGF (PeproTech, Rocky Hill, NJ, United States). All dog owners provided written informed consent for the current study, and all experimental procedures were carried out following the guidelines of the Institute Animal Care and Use Committee of Tokyo University of Agriculture and Technology (Approval number: 0020007). Table 1 includes sample information.

Abugomaa A., Elbadawy M., Ishihara Y., Yamamoto H., Kaneda M., Yamawaki H., Shinohara Y., Usui T, & Sasaki K. (2023). Anti-cancer activity of Chaga mushroom (Inonotus obliquus) against dog bladder cancer organoids. Frontiers in Pharmacology, 14, 1159516.

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Isolation and Culture of Mouse Intestinal Organoids

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Small intestinal crypts were derived from wildtype C57BL/6 mice using a slightly modified protocol from reference^{51 (link)}. In brief, collected small intestines were opened longitudinally and the majority of villi removed by gentle scraping with a coverslip. The tissue was cut to 3–5 mm pieces, washed five times with cold PBS, and vigorous shaking. After 30 min incubation on ice with 2 mM EDTA/ PBS, the remaining villi were removed with short shaking and tissue pieces were incubated for additional 30 min in 5 mM EDTA/PBS on ice. After another short shake, crypts were passed through a 40-μm cell strainer and pelleted at 425×g 1500 rpm at 4 °C for 10 min. The pellet was washed with cold PBS and 100–200 crypts were suspended in 50 μl Red-phenol-free Matrigel (BD Biosciences) droplets. After polymerization, complete medium containing advanced DMEM/F12 (Sigma), 2 mM Glutamax (Invitrogen), 10 mM HEPES (Gibco), 100 U/ml penicillin/streptomycin (Invitrogen), 1 mM N-acetyl-cysteine (Sigma), 1× B27 supplement (Invitrogen), 1× N₂ supplement (Invitrogen), 50 ng/ml mouse EGF (Peprotech), 100 ng/ml mouse Noggin (Peprotech), and 10% human R-spondin-1-conditioned medium from R-spondin-1-transfected HEK293T cells (Cultrex) was added to the cultures. Medium was changed every 3 days and organoids passaged after 7–10 days.

Fellows R., Denizot J., Stellato C., Cuomo A., Jain P., Stoyanova E., Balázsi S., Hajnády Z., Liebert A., Kazakevych J., Blackburn H., Corrêa R.O., Fachi J.L., Sato F.T., Ribeiro W.R., Ferreira C.M., Perée H., Spagnuolo M., Mattiuz R., Matolcsi C., Guedes J., Clark J., Veldhoen M., Bonaldi T., Vinolo M.A, & Varga-Weisz P. (2018). Microbiota derived short chain fatty acids promote histone crotonylation in the colon through histone deacetylases. Nature Communications, 9, 105.

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Intestinal Organoid Culture Reagents

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Fetal bovine serum (FBS), DMEM, Minimum Essential Medium Eagle (MEM), CDCA, n-acetylcysteine (NAC), [Leu]15-Gastrin 1, NT peptide and β-actin antibody were from Sigma-Aldrich (St. Louis, MO). GW4064 was from Tocris (Minneapolis, MN). The phospho ERK1/2 and ERK1/2 antibodies were from Cell signaling (Danvers, MA). FXR and NTR1 antibodies were from Santa Cruz (Dallas, TX). Noggin-conditioned medium was purchased from U-Protein Express BV (Netherlands). Advanced DMEM/F12 medium, OptiMEM Reduced Serum Medium, growth factor–reduced Matrigel, B-27 Supplement, N-2 Supplement, HEPES, GlutaMAX and Zeocin were from ThermoFisher (Grand Island, NY). Mouse EGF was from PeproTech (Rocky Hill, NJ).

Li J., Song J., Yan B., Weiss H.L., Weiss L.T., Gao T, & Evers B.M. (2021). Neurotensin differentially regulates bile acid metabolism and intestinal FXR-bile acid transporter axis in response to nutrient abundance. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 35(5), e21371.

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Organoid Generation from Colorectal Tissues

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To generate organoids, human colorectal tissues were cultured with modified ISCs media as described previously [9 (link)]. The components were as follows: Advanced Dulbecco's Modified Eagle's Medium (DMEM) with 50% Wnt, Noggin, and R-Spondin conditioned medium; GlutaMax; B-27 supplement; 100 μg/mL Primocin (Invitrogen, Carlsbad, CA, USA); 1 mM N-acetyl-L-cysteine; 10 mM Nicotinamide (Sigma-Aldrich, St. Louis, MO, USA); 50 ng/mL mouse EGF (PeproTech, Inc., Rocky Hill, NJ, USA); 500 nM A83-01 (Adooq Bioscience, Irvine, CA, USA); 3 μM SB202190; 10 μM Y-27632 (Cayman, Ann Arbor, MI, USA). Anticancer drugs were as follows: 5-fluorouracil (5-FU) (WAKO, Tokyo, Japan); Irinotecan (LC Laboratories, Woburn, MA, USA). Antibody sources were as follows: E-cadherin (R&D System, Minneapolis, MN, USA); MUC2 (Gene Tex, Irvine, CA, USA); vimentin (Sigma-Aldrich); α-smooth muscle actin (SMA) (DAKO, Glostrup, Denmark); LGR5 (Abgent, San Diego, CA, USA); CD44 (Bethyl Laboratories, Montgomery, TX, USA). Secondary antibodies were as follows: Alexa Fluor 488 goat anti-mouse IgG; Alexa Fluor 488 goat anti-rabbit IgG; Alexa Fluor 568 goat anti-rabbit IgG; Alexa Fluor 488 donkey anti-goat IgG (Invitrogen).

Usui T., Sakurai M., Enjoji S., Kawasaki H., Umata K., Ohama T., Fujiwara N., Yabe R., Tsuji S., Yamawaki H., Hazama S., Takenouchi H., Nakajima M., Tsunedomi R., Suzuki N., Nagano H, & Sato K. (2016). Establishment of a Novel Model for Anticancer Drug Resistance in Three-Dimensional Primary Culture of Tumor Microenvironment. Stem Cells International, 2016, 7053872.

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