Recombinant murine egf

Manufactured by Thermo Fisher Scientific

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Recombinant murine EGF is a protein that acts as a growth factor, specifically for mouse cells. It is a laboratory reagent used in cell culture and research applications.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions) N acetylcysteine, by Merck Group (6 mentions) Advanced dmem f12, by Thermo Fisher Scientific (5 mentions) Matrigel, by Corning (5 mentions) Recombinant murine noggin, by Thermo Fisher Scientific (4 mentions) Hepes, by Thermo Fisher Scientific (4 mentions) L glutamine, by Thermo Fisher Scientific (3 mentions) B27 supplement, by Thermo Fisher Scientific (3 mentions) Glutamax, by Thermo Fisher Scientific (3 mentions) N acetyl cysteine (nac), by Thermo Fisher Scientific (2 mentions) Hepes, by HiMedia (2 mentions) Hepes solution, by Merck Group (2 mentions) 27g syringe, by BD (2 mentions) 4oh tamoxifen, by Cayman Chemical (2 mentions) Recombinant human r spondin1, by R&D Systems (2 mentions) Dmem f12, by Thermo Fisher Scientific (2 mentions) Recombinant human r spondin 1, by Thermo Fisher Scientific (2 mentions) Recombinant human fgf basic, by Thermo Fisher Scientific (2 mentions) Recombinant murine fgf basic, by Thermo Fisher Scientific (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions)

Spelling variants of this product

Recombinant EGF (35 citations) Murine EGF (30 citations)

28 protocols using recombinant murine egf

Anthrax Toxin Exposure in Organoids

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Organoids were seeded in 96-well plates with transparent bottom and opaque walls in 5µl Matrigel domes. After two days in organoid media, organoids were grown for an additional 3 days in differentiation media: DMEM/F12 supplemented with 20% murine R-Spondin1 supernatant, 10% murine Noggin supernatant, 50 ng/mL recombinant murine EGF (Fisher Scientific), 1X P/S, 1X L-Glu, 10 mM HEPES (HiMedia), 1X N2 (Life Technologies), 1X B27 (Life Technologies), and 1 mM Nacetylcysteine (Fisher Scientific). 5 µM DAPT was added for the last 24 hours. Differentiated organoids were then treated with 16µg/ml PA and 8µg/ml LFn-FlaA or 0.1 µg/ml LFn-PrgJ in differentiation media containing 10µg/ml propidium iodide for 4 hours.
Total lysis wells were treated with 1%Triton.

Naseer N., Bauer R., Zhang J., Brodsky I.E., Rauch I., & Shin S. (2021). Salmonella Typhimurium induces NAIP/NLRC4- and NLRP3/ASC-independent, caspase-1/4-dependent inflammasome activation in human intestinal epithelial cells.

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Inflammasome Expression in Spheroid Cultures

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To analyze inflammasome expression under different media conditions, spheroids were cultured in differentiation medium: DMEM/F12 supplemented with 20% supernatant from R-Spondin1 expressing L-cells, 10% supernatant from Noggin expressing cells, 50 ng/mL recombinant murine EGF (Fisher Scientific), 1X P/S, 1X L-Glu, 10 mM HEPES (HiMedia), 1X N2 (Life Technologies), 1X B27 (Life Technologies), 1 mM N-acetylcysteine (Fisher Scientific), and 5 µM DAPT. After 4 days of incubation at 37°C, Matrigel domes were dissolved in PBS-EDTA (5 mM) for 1 hour at 4°C on an orbital shaker. After centrifugation at 300 × g for 5 minutes at 4°C, the cell pellet was resuspended in TRIzol TM to analyze mRNA expression.

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EGF-mediated EGFR Stimulation in Mice

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S/A;fTg/0 (Cont.) and A/A;fTg/0;Cre^Hep/0 (A/A^Hep) mice have been previously described (Choi et al., 2017 (link)). The animals were housed with 12-h light and 12-h dark cycles, and provided with standard rodent chow (Purina; Cargill Inc., USA) and water ad libitum. All animal care and procedures were conducted according to the protocols and guidelines approved by the University of Ulsan Animal Care and Use Committee (UOUACUC) (No. SHB-17-010).
The Cont. and A/A^Hep mice fasted for 12 h prior to in vivo EGF-mediated EGFR stimulation. After anesthesia was induced, the mice received 0.9% saline or 0.9% saline containing recombinant murine EGF (0.5 μg/g of body weight; PeproTech, Korea) via the vena cava. The liver was removed 5 min after this injection. The specimens were frozen and stored at –80°C until homogenization.

Kim M.J., Choi W.G., Ahn K.J., Chae I.G., Yu R, & Back S.H. (2020). Reduced EGFR Level in eIF2α Phosphorylation-Deficient Hepatocytes Is Responsible for Susceptibility to Oxidative Stress. Molecules and Cells, 43(3), 264-275.

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Generation and Manipulation of Mouse Jejunal Enteroids

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Mouse jejunal enteroids were generated following established protocols (Nalapareddy et al, 2017 (link)). Briefly, crypt domains from small intestinal tissue were isolated, added to 40 µl of Matrigel (Corning) membrane matrix, suspended as a single 3D Matrigel dome within a 24‐well plate, and provided 400 µl mouse enteroid media (Advanced DMEM/F‐12 (Gibco), L‐Glutamine (Gibco), Penicillin/Streptomycin (Gibco), N‐2 (Gibco), B‐27 (Gibco), 10 mM HEPES solution (Millipore‐Sigma), 50 ng/ml recombinant murine EGF (Peprotech), and R‐Spondin/Noggin conditioned media generated in‐house. Enteroids were passaged every 4–7 days via a 27G syringe (BD). CRE‐driven Bmal1 exon 4 or Rac1 exon 1 excisions were initiated in vitro by adding 1 µM 4‐OH tamoxifen (Cayman) for 24‐h to Bmal1^f/f‐EsrCRE or Rac1^f/f;CAGGCre‐ER™ enteroids. All samples were maintained in cell culture incubators set at 37°C and 5% CO₂. Enteroids isolated from separate mice were considered biological replicate samples.

Rosselot A.E., Park M., Kim M., Matsu‐Ura T., Wu G., Flores D.E., Subramanian K.R., Lee S., Sundaram N., Broda T.R., McCauley H.A., Hawkins J.A., Chetal K., Salomonis N., Shroyer N.F., Helmrath M.A., Wells J.M., Hogenesch J.B., Moore S.R, & Hong C.I. (2021). Ontogeny and function of the circadian clock in intestinal organoids. The EMBO Journal, 41(2), e106973.

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Culturing Human Intestinal Organoids

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A thin film of liquid type I collagen (Advanced BioMatrix, San Diego, CA) was plated onto the bottom surface of 48-well Nucleon Delta-treated cell culture plate (Thermo Scientific, Waltham, MA) at a concentration of 100 ng/mL, incubated for 30 minutes, and then removed by aspiration. Human crypts were plated at a density of 500 crypts per well. These were grown as monolayers in Basic Medium with antibiotic-antimycotic (Invitrogen), 1 mM N-Acetylcysteine (Sigma), 100 ng/ml recombinant murine Noggin (PeproTech), 50 ng/ml recombinant murine EGF (PeproTech), 1xN2 supplement (Invitrogen), 1xB27 supplement (Invitrogen), 1 μg/ml recombinant human R-spondin 1 (R&D Systems, Minneapolis, MN), 10 μM Y-27632 inhibitor (Stemgent), 1 mM recombinant human Jagged-1 (R&D Systems), 5 μM CHIR99021 (GSK-3β inhibitor) (Stemolecule). Alternatively, crypts were suspended within Matrigel at a concentration of 100 crypts per 25 μl of Matrigel and plated in 3D on the 48-well Nucleon Delta-treated cell culture plate.

Rouch J.D., Scott A., Lei N.Y., Solorzano-Vargas R.S., Wang J., Hanson E.M., Kobayashi M., Lewis M., Stelzner M.G., Dunn J.C., Eckmann L, & Martín M.G. (2016). Development of Functional Microfold (M) Cells from Intestinal Stem Cells in Primary Human Enteroids. PLoS ONE, 11(1), e0148216.

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EGF Stimulation of Confluent Cells

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Confluent cells were stimulated by adding 10 ng/μL EGF (Epidermal Growth Factor) (Recombinant murine EGF, PeproTech, Rocky Hill, NJ, USA) for 5 min into the medium. After incubation time, medium was immediately removed and cells were washed with ice cold PBS. Total cell lysates were analyzed via Western Blotting.

Catalano A., Adlesic M., Kaltenbacher T., Klar R.F., Albers J., Seidel P., Brandt L.P., Hejhal T., Busenhart P., Röhner N., Zodel K., Fritsch K., Wild P.J., Duyster J., Fritsch R., Brummer T, & Frew I.J. (2021). Sensitivity and Resistance of Oncogenic RAS-Driven Tumors to Dual MEK and ERK Inhibition. Cancers, 13(8), 1852.

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EGFR Signaling Pathway Analysis

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Antibodies and reagents were purchased as follows: mAb-2E9 (Abcam ab8465, RRID: AB_2096462), Anti-EGFR Affibody® Molecule (Abcam ab95116; RRID: AB_11156238), Anti-EGFR (D38B1, Cell Signaling Technology (CST) 4267; RRID: AB_2246311), Anti-Phospho-Akt (Ser473, D9E, CST 4060; RRID: AB_2315049), Anti-beta-Actin Monoclonal Antibody, HRP Conjugated (13E5 CST 5125; RRID: AB_1903890), Anti-Phospho-EGF Receptor (Tyr1068, D7A5 CST 3777; RRID: AB_2096270), Anti-EGFR (R & D Systems, AF231; RRID: AB_355220), Anti-EGFR (phospho Y992, EM-12, Abcam ab81440; RRID: AB_1658463), Anti-mouse IgG-HRP (Jackson ImmunoResearch 715-035-150; RRID: AB_2340770), Anti-rabbit IgG-HRP (Jackson ImmunoResearch 711-035-152; RRID: AB_10015282), Anti goat IgG-HRP (Jackson ImmunoResearch 705-035-147; RRID: AB_2313587), Anti-Rabbit-HRP (Dako P044801, RRID: AB_2617138), Recombinant Murine EGF (Peprotech, 315-09), Recombinant Murine IL-3 (Peprotech, 213-13), Erlotinib Hydrochloride (Biovision, 1558-100), Lapatinib Ditosylate (Biovision, 1642-25), 4% Paraformaldehyde (EM Grade, Electron Microscopy Service (EMS), 157-4), 25% Glutaraldehyde (Grade I, Sigma G5882), FluoSpheres™ (carboxylate modified, 0.1 μm, infrared (715/755), Invitrogen F8799).

Iyer R.S., Needham S.R., Galdadas I., Davis B.M., Roberts S.K., Man R.C., Zanetti-Domingues L.C., Clarke D.T., Fruhwirth G.O., Parker P.J., Rolfe D.J., Gervasio F.L, & Martin-Fernandez M.L. (2024). Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers. Nature Communications, 15, 2130.

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Intestinal Organoid Isolation and Culture

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The following protocol was used with minor modifications, based on previously published methods (103 (link)). The intestinal segment was dissected into small pieces and incubated in dissociation solution (PBS supplemented with 2 mM EDTA and 10 μM Rho kinase inhibitor Y-27632) for 1 hour at 4°C with agitation. Next, intestinal tissue pieces were shaken, strained, and centrifuged; pellets were resuspended in Matrigel (Corning Inc.); and ENR medium was added. ENR is a basal medium supplemented with recombinant murine EGF (50 ng/ml) (PeproTech), recombinant murine Noggin (50 ng/ml) (PeproTech), 1 mM N-acetylcysteine (Sigma-Aldrich), and 20% (v/v) of R-Spondin–conditioned medium (Cultrex Rspo1-expressing cells, Trevigen). The basal medium is advanced Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco) supplemented with 3 mM l-glutamine (Thermo Fisher Scientific), Primocin (100 μg/ml) (InvivoGen), and 10 mM Hepes (Sigma-Aldrich). For tumor-derived organoids, dissected polyps were prepared as described above, with an additional 1-hour incubation in digestion buffer [2.5% FBS, P/S (1 μg/ml), type IV collagenase (200 U/ml), and type II dispase in DMEM (125 μg/ml)].

Chun S.K., Fortin B.M., Fellows R.C., Habowski A.N., Verlande A., Song W.A., Mahieu A.L., Lefebvre A.E., Sterrenberg J.N., Velez L.M., Digman M.A., Edwards R.A., Pannunzio N.R., Seldin M.M., Waterman M.L, & Masri S. (2022). Disruption of the circadian clock drives Apc loss of heterozygosity to accelerate colorectal cancer. Science Advances, 8(31), eabo2389.

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Colon Organoid Culture Protocol

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Colon crypts were resuspended in ice-cold Matrigel (Corning, Cat. 356231) and allowed to solidify at 37°C, then were supplied with culture medium (750 ng/ml recombinant human Rspondin-1 (lab-made), 100 ng/ml recombinant murine Noggin (PeproTech, Cat. 250-38), 50 ng/ml recombinant murine EGF (PeproTech, Cat. 315-09), 10 μM CHIR99021 (Tocris, Cat. 4423/10), 100 ng/ml recombinant murine Wnt-3a (PeproTech, Cat. 315-20), N2 (Thermo Fisher Scientific, Cat. 15410294), B27 (Thermo Fisher Scientific, Cat. 11500446), 1X Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. 15140122) in Advanced DMEM/F12 (ThermoFisherScientific, Cat. 11550446)). Organoids were cultured for 7 days, passaged via re-suspension in ice-cold PBS, then cultured for an additional 3 days before being collected in QIAzol (Qiagen, Cat. 79306) for RNA isolation.

Širvinskas D., Omrani O., Lu J., Rasa M., Krepelova A., Adam L., Kaeppel S., Sommer F, & Neri F. (2022). Single-cell atlas of the aging mouse colon. iScience, 25(5), 104202.

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Murine Small Intestinal Organoid Culture

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Small intestinal organoids were established and maintained as described (Sato et al., 2009 (link)), starting from isolated crypts collected from the entire length of the small intestine of B6 mice. The basic culture medium (advanced DMEM/F12, penicillin/streptomycin, 10 mM HEPES, 1x GlutaMAX, 1x N-2 supplement, 1x B-27 supplement (all from Gibco, Thermo Fisher Scientific Cat. # 12634010, 400–109, 15630080, 35050061, 17502001 and 17504044, respectively), 1 mM N-acetylcysteine (MilliporeSigma Cat. # 106425)), was supplemented with 50 ng/ml recombinant murine EGF (Peprotech Cat. # 315–09), and 1% v/v Noggin CM to obtain EN medium. Purified RSPO recombinant proteins were added to the basic culture medium as indicated. Organoids were cultured in matrigel droplets (Corning CellBIND, Corning Cat. # 3300, 3335, 3292). The medium was refreshed 3 and 6 days after splitting. Images were captured 8 days after splitting using an EVOS M5000 imaging system (Thermo Fisher Scientific).

Dubey R., van Kerkhof P., Jordens I., Malinauskas T., Pusapati G.V., McKenna J.K., Li D., Carette J.E., Ho M., Siebold C., Maurice M., Lebensohn A.M, & Rohatgi R. (2020). R-spondins engage heparan sulfate proteoglycans to potentiate WNT signaling. eLife, 9, e54469.

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