Mouse recombinant noggin

Manufactured by Thermo Fisher Scientific

Sourced in United States, Switzerland

Thermo Fisher Scientific's Mouse Recombinant Noggin is a purified protein that functions as a bone morphogenetic protein (BMP) antagonist. Noggin binds to and inhibits the activity of various BMPs, thereby regulating developmental processes such as embryonic patterning, skeletal morphogenesis, and neural induction.

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Noggin (258 citations) Recombinant Noggin (21 citations) Mouse Noggin (8 citations)

15 protocols using mouse recombinant noggin

Stem Cell Culture with Signaling Modulators

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Hoechst 33342, Ko143 and YHO-13177 were purchased from Med Chem Express (Monmouth Junction, NJ, USA). Recombinant human R-spondin 1 and recombinant mouse noggin were obtained from Pepro Tech Inc. (Rocky Hill, NJ, USA). Mouse recombinant EGF, advanced DMEM/F12, B-27 supplement and N-2 supplement were supplied by Invitrogen (Carlsbad, CA, USA). Matrigel (GFR, phenol-free) was bought from Corning Inc. (Bedford, MA, USA). Anti-BCRP antibody and goat anti-rabbit antibody were purchased from Abcam (Cambridge, UK). Trizol, PrimeScript™ RT reagent Kit and SYBR^® Premix Ex Taq™ II MIX were supplied by Takara (Dalian, China).

Zhang L., Zhao J., Liang C., Liu M., Xu F, & Wang X. (2017). A novel biosensor based on intestinal 3D organoids for detecting the function of BCRP. Drug Delivery, 24(1), 1453-1459.

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Kidney Organoids from Tubular Fragments

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Kidneys were isolated and tubular fragments were isolated by collagenase digestion (C9407, Sigma) for 30 min at 1 mg ml⁻¹. Fragments were seeded in growth factor-reduced Matrigel (Corning) and cultured in medium (DMEM/F12 supplemented with 1% penicillin/streptomycin, HEPES, GlutaMAX), with 2% B27 supplement (Gibco), recombinant mouse noggin (50 ng ml⁻¹, Peprotech), 10% Rspo1 (Millipore-Sigma, SCM104), EGF (50 ng ml⁻¹, Peprotech), FGF-10 (100 ng ml⁻¹, Peprotech), N-acetylcysteine (1.25 mM, Sigma), A8301 (5 µM, Tocris Bioscience) and primocine (0.1 mg ml⁻¹, Invivogen). After 2 weeks, GEKOs were cultured using DMEM/F12 supplemented with 1% P/S (penicillin/streptomycin), 10% FBS^{46 (link)}.

Perelli L., Carbone F., Zhang L., Huang J.K., Le C., Khan H., Citron F., Del Poggetto E., Gutschner T., Tomihara H., Soeung M., Minelli R., Srinivasan S., Peoples M., Lam T.N., Lundgren S., Xia R., Zhu C., Mohamed A.M., Zhang J., Sircar K., Sgambato A., Gao J., Jonasch E., Draetta G.F., Futreal A., Bakouny Z., Van Allen E.M., Choueiri T., Signoretti S., Msaouel P., Litchfield K., Turajlic S., Wang L., Chen Y.B., Di Natale R.G., Hakimi A.A., Giuliani V., Heffernan T.P., Viale A., Bristow C.A., Tannir N.M., Carugo A, & Genovese G. (2023). Interferon signaling promotes tolerance to chromosomal instability during metastatic evolution in renal cancer. Nature Cancer, 4(7), 984-1000.

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Isolation and Culture of Glomerular Epithelial Cells

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Kidneys were isolated and tubular fragments were collected by collagenase digestion (C9407, Sigma) for 30 min at 0.5 mg ml⁻¹ at 37 °C and 5% CO₂. Fragments were centrifuged for 5 min at 150g, washed and resuspended in appropriate medium (DMEM/F12 supplemented with 1% penicillin/streptomycin, HEPES, GlutaMAX), with 1.5% B27 supplement (Gibco), recombinant mouse noggin (50 ng ml⁻¹, Peprotech), 10% Rspo1 (Millipore-Sigma, SCM104), EGF (50 ng ml⁻¹, Peprotech), FGF-10 (100 ng ml⁻¹, Peprotech), N-acetylcysteine (1.25 mM, Sigma), A8301 (5 µM, Tocris Bioscience) and primocine (0.1 mg ml⁻¹, Invivogen). After five passages, GEKTCs were cultured using DMEM/F12 supplemented with 1% P/S, 10% FBS.

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Intestinal Organoid Culture Protocol

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N2 and B27 supplements, GlutaMAX, Advanced DMEM/F12 base media, 5-ethynyl-2′-deoxyuridine (EdU) kit and α-goat-Alexa Fluor 488 were purchased from Invitrogen (Carlsbad, CA). Y27632 Rock inhibitor, HEPES buffer, N-acetylcysteine (NAC), ethylenediaminetetraacetic acid (EDTA, 0.5 M, pH 8.0), bis-Benzimide (Hoescht 33342), and α–rabbit-Cy3 were obtained from Sigma-Aldrich (St. Louis, MO). Cell-culture-grade bovine serum albumin (BSA), dithiothreitol (DTT) and a pulse vortex-mixer were purchased from Thermo-Fisher (Fairlawn, NJ). Recombinant mouse Wnt-3a, recombinant human R-Spondin1 and recombinant mouse EGF were acquired from R&D Systems (Minneapolis, MN). Growth-factor reduced Matrigel was obtained from BD Biosciences (Bedford, MA). Recombinant mouse Noggin was purchased from Peprotech (Rocky Hill, NJ). Sylgard 184 silicone elastomer kit was procured from Dow Corning (Midland, MI). EPON epoxy resin 1002-F (fusion solids) was purchased from Miller Stephenson Chemical Co. (Sylmar, CA). Primary antibodies α-mucin2 and α-chromogranin A were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Ahmad A.A., Wang Y., Gracz A.D., Sims C.E., Magness S.T, & Allbritton N.L. (2014). Optimization of 3-D organotypic primary colonic cultures for organ-on-chip applications. Journal of Biological Engineering, 8, 9.

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In Vivo Administration of Small Molecule Inhibitors

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E6201 was obtained from Spirita Oncology. LDN-193189 was purchased from Sigma-Aldrich. Trametinib and Buparlisib were purchased from MedKoo Biosciences. Recombinant mouse noggin was from Preprotech, and mouse follistatin was from Shenandoah Biotechnology. For in vivo administration, E6201 was supplied by Spirita Oncology in lyophilized form, pre-weighted in sealed vials, and reconstituted with sterile water for injection, yielding a final concentration of 6 mg/mL in 30% Captisol. Vehicle control solution was prepared by dissolving Captisol in sterile water for injection. The drug was dissolved freshly before each injection. The drug was administered by intraperitoneal injections, using 27 Gauge needles fitted to 0.5 mL syringes.

Fortin J., Tian R., Zarrabi I., Hill G., Williams E., Sanchez-Duffhues G., Thorikay M., Ramachandran P., Siddaway R., Wong J.F., Wu A., Apuzzo L.N., Haight J., You-Ten A., Snow B.E., Wakeham A., Goldhamer D.J., Schramek D., Bullock A.N., Dijke P.T., Hawkins C, & Mak T.W. (2020). Mutant ACVR1 Arrests Glial Cell Differentiation to Drive Tumorigenesis in Pediatric Gliomas. Cancer Cell, 37(3), 308-323.e12.

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Enteroid Culture and Quantification

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Approximately 100 isolated jejunal crypts were resuspended in 20 μl Matrigel (Corning) and placed in 48 well tissue culture plates. After polymerization of the Matrigel, 250 μl of media was added per well. The media consisted of Advanced DMEM/F12 (Invitrogen) containing growth factors (50 ng/ml recombinant mouse EGF (R&D Systems), 500 ng/ml R-spondin 1 (R&D Systems), 100 ng/ml recombinant mouse Noggin (Peprotech), 1x N2 supplement (Gibco), 1x B27 (Gibco), 10 µM HEPES (Gibco), 1x Glutamax (Gibco), and 500 µg/ml penicillin-streptomycin (Gibco)). Media was changed every other day. Enteroids were imaged and counted one day after plating to determine % plating efficiency (# enteroids growing/#crypts plated) using an inverted Olympus IX83 microscope.

Sheahan B.J., Theriot C.M., Cortes J.E, & Dekaney C.M. (2022). Prolonged oral antimicrobial administration prevents doxorubicin-induced loss of active intestinal stem cells. Gut Microbes, 14(1), 2018898.

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Generating Intestinal Organoids from Mouse Crypts

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SI organoids were derived from Hvem^/− or littermate Hvem^+/+ mice. The proximal SI was cut into 5-mm segments and incubated in 4°C 2 mM EDTA in PBS for 5 min and washed by pipetting. The segments were incubated in 2 mM EDTA in PBS for 30 min at 4°C, and crypts were isolated by pipetting with cold HBSS. Dissociated crypts were passed through a 70-μm cell strainer and pelleted by centrifuge at 600 rpm for 3 min at 4°C. The crypts were resuspended in Advanced DMEM/F12 medium (Thermo Fisher Scientific); the number of crypts was counted, and they were resuspended in Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning). The crypts were plated in a 24-well plate with organoid growth medium supplemented with 100 μg/ml penicillin, 100 U/ml streptomycin, 2 mM Glutamax, 1×N-2 supplement, 1×B27 supplement, 10 mM HEPES (Thermo Fisher Scientific), 1 mM N-acetylcysteine (Sigma-Aldrich), 100 ng/ml recombinant mouse Noggin (Peprotech), 50 ng/ml recombinant mouse EGF (BioLegend), 500 ng/ml recombinant human R-spondin 1 (Peprotech). Media were changed every 2 days.

Seo G.Y., Takahashi D., Wang Q., Mikulski Z., Chen A., Chou T.F., Marcovecchio P., McArdle S., Sethi A., Shui J.W., Takahashi M., Surh C.D., Cheroutre H, & Kronenberg M. (2022). Epithelial HVEM maintains intraepithelial T cell survival and contributes to host protection. Science immunology, 7(73), eabm6931.

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Maintenance of ABCG2-positive hPSC-Derived LSCs

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For maintenance of the ABCG2-positive population, CnT-30 was replaced with CnT-07 (CELLnTEC Advanced Cell Systems AG, Bern, Switzerland) supplemented with ENRC (50 ng/ml mouse recombinant epidermal growth factor (EGF, Invitrogen), 100 ng/ml mouse recombinant Noggin, 1 μg/ml human recombinant R-spondin (both from PeproTech), and 3 μM CHIR-99021 (Stemgent)) at d10–11 of the standard CnT-30 differentiation protocol. New medium was introduced directly to the adherent cultures; alternatively, the hPSC-LSCs were concomitantly passaged onto fresh LN-521/Col IV-coated wells at a density of 1 000 cells/cm² in the new medium. The cells were thereafter cultured following the standard feeding regimen. After the emergence of ABCG2-positive colonies in approximately 7–10 days, further expansion of the ABCG2-positive hPSC-LSCs in the ENRC medium was carried out by passaging subconfluent cultures onto fresh LN-521/Col IV-coated matrices at a density of 1 000 cells/cm². Batches of the passaged cells were also cryopreserved following our routine cryopreservation protocol [24 (link), 25 ]. Throughout the article, the term “ENRC maintenance condition” refers to the novel CnT-07-based, ENRC-supplemented culture protocol described in this chapter.

Vattulainen M., Ilmarinen T., Koivusalo L., Viiri K., Hongisto H, & Skottman H. (2019). Modulation of Wnt/BMP pathways during corneal differentiation of hPSC maintains ABCG2-positive LSC population that demonstrates increased regenerative potential. Stem Cell Research & Therapy, 10, 236.

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Isolation and Culture of Mouse Lung Organoids

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Mouse lungs were removed, flushed, cut into 5-mm³ pieces and incubated with digestion buffer containing 1.0 mg ml⁻¹ collagenase I (Gibco, 17100–017) and 0.5 mg ml⁻¹ collagenase IV (Gibco, 17104–019) in DMEM/F12 (Gibco, C11330500BT) in gentleMACS C tubes (Miltenyi Biotec, 130-096-334). After dissociation by the gentleMACS dissociator, samples were filtered through 70-μm filters, collected by centrifugation and resuspended in ice-cold Matrigel (BD, 354230) at a ratio of 1:20 (vol:vol). The basic culture medium for mouse lung organoids was slightly modified from a previous report^{43 (link)}, where DMEM/F12 was supplemented with penicillin/streptomycin (Gibco, 15140–122), 2 mM GlutaMAX (Peprotech, 35050–061), 1× B27 (Gibco, A3582801), 1× N2 (Gibco, 17502048), 10 nM gastrin I (Peprotech, 1003377), 1 mM N-acetylcysteine (Sigma, A9165) and 10 mM nicotinamide (Sigma, N0636). The following growth factors were used: 50 ng ml⁻¹ mouse recombinant epidermal growth factor (Peprotech, AF-100-15-1000), 100 ng ml⁻¹ mouse recombinant noggin (Peprotech, 120-10C-250), 500 ng ml⁻¹ mouse recombinant FGF10 (Peprotech, 100-26-1000), 125 ng ml⁻¹ R-spondin-1 (Peprotech, 120-38-1000), 10% Wnt-3A conditioned medium and 500 nM A83–01 (Peprotech, 9094360). The medium was changed every 3 d. Organoids were passaged by mechanical dissociation in TrypLE (Gibco, 12605–028) every 5–7 d.

Na F., Pan X., Chen J., Chen X., Wang M., Chi P., You L., Zhang L., Zhong A., Zhao L., Dai S., Zhang M., Wang Y., Wang B., Zheng J., Wang Y., Xu J., Wang J., Wu B., Chen M., Liu H., Xue J., Huang M., Gong Y., Zhu J., Zhou L., Zhang Y., Yu M., Tian P., Fan M., Lu Z., Xue Z., Zhao Y., Yang H., Zhao C., Wang Y., Han J., Yang S., Xie D., Chen L., Zhong Q., Zeng M., Lowe S.W., Lu Y., Liu Y., Wei Y, & Chen C. (2022). KMT2C deficiency promotes small cell lung cancer metastasis through DNMT3A-mediated epigenetic reprogramming. Nature cancer, 3(6), 753-767.

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Genetically Engineered Colon and Gastric Organoids

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For organoid experiments, normal colon organoids with genetic engineering of TP53 KO and RB1 KO, and gastric organoids with TP53 KO were used in this experiment as previously reported (16 (link)). Culture conditions were as previously described, with Advanced Dulbecco's Modified Eagle Medium/F12 supplemented with penicillin/streptomycin, 10 mmol/L HEPES, 2 mmol/L Gluta-MAX, 1 × B27 (Thermo Fisher Scientific), 10 nmol/L gastrin I (MilliporeSigma), and 1 mmol/L N-acetylcysteine (FUJIFILM Wako Pure Chemical Corporation). Complete medium was prepared by supplementing the basal culture medium with the following niche factors: 50 ng/mL mouse recombinant EGF (Thermo Fisher Scientific), 50 ng/mL human recombinant FGF2 (PeproTech), 100 ng/mL human recombinant IGF1 (BioLegend), 100 ng/mL mouse recombinant noggin (PeproTech), 1 μg/mL recombinant human R-spondin-1 (Bio-Techne), 25% Afamin-Wnt-3A serum-free conditioned medium, and 500 nmol/L A83-01 (Tocris Bioscience). For organoid experiments, N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT; MilliporeSigma, 10 μmol/L) was added on day 1. EGF/FGF2/IGF1 were removed from the culture media on day 5, and the organoids were collected on day 10, fixed with 4% paraformaldehyde, and immunostained using mouse antisynaptophysin.

Yachida S., Totoki Y., Noë M., Nakatani Y., Horie M., Kawasaki K., Nakamura H., Saito-Adachi M., Suzuki M., Takai E., Hama N., Higuchi R., Hirono S., Shiba S., Kato M., Furukawa E., Arai Y., Rokutan H., Hashimoto T., Mitsunaga S., Kanda M., Tanaka H., Takata S., Shimomura A., Oshima M., Hackeng W.M., Okumura T., Okano K., Yamamoto M., Yamaue H., Morizane C., Arihiro K., Furukawa T., Sato T., Kiyono T., Brosens L.A., Wood L.D., Hruban R.H, & Shibata T. (2022). Comprehensive Genomic Profiling of Neuroendocrine Carcinomas of the Gastrointestinal System. Cancer Discovery, 12(3), 692-711.

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