Mouse gm csf

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Mouse GM-CSF is a recombinant cytokine that stimulates the production and function of granulocytes and macrophages. It is commonly used in cell culture applications to study the differentiation and proliferation of myeloid cells.

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GM-CSF (1367 citations) Recombinant mouse GM-CSF (103 citations) Recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF) (16 citations) Recombinant mouse GM-CSF and IL-4 (7 citations) GM-CSF Mouse ELISA Kit (5 citations) Mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) (5 citations) Mouse recombinant (rm) granulocyte-macrophage colony-stimulating factor (GM-CSF) (4 citations) Mouse GM-CSF and IL-4 (2 citations) GM-CSF Mouse Uncoated ELISA Kit (2 citations)

36 protocols using mouse gm csf

Generation of Bone Marrow-Derived Monocytic Myeloid-Derived Suppressor Cells

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The protocol for BM-Mo-MDSC preparation was modified from that used in a previous report. Femurs were removed from C57BL/6 mice and the BM was flushed. Red blood cells were lysed with ammonium chloride. BM cells were adjusted to a concentration of 1 × 10⁶/ml in 10% FBS-containing RPMI1640 medium supplemented with 20 ng/ml mouse GM-CSF, 40 ng/ml mouse IL-6 and 2 ng/ml mouse IFN-γ (Peprotech). Cells were maintained at 37 °C in a 5% CO₂- and 3% O₂-humidified atmosphere for 4–5 days. Non-adherent and loosely adherent cells were collected by gentle pipetting. In some experiments, BM-Mo-MDSCs were treated with flavopiridol HCl (Selleckchem) or HLM (Tocris Bioscience) along with GM-CSF, IL-6, and IFN-γ on day 4, and collected 24 h later.

Okuma A., Hanyu A., Watanabe S, & Hara E. (2017). p16Ink4a and p21Cip1/Waf1 promote tumour growth by enhancing myeloid-derived suppressor cells chemotaxis. Nature Communications, 8, 2050.

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Dendritic Cell Differentiation and Activation

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Bone marrow cells were collected from the tibia and femur of wildtype C57BL/6 J mice. Cells were cultured for 3 d at a density of 2 × 10⁵ cells/ml with mouse GM-CSF (PeproTech, 20 ng/ml), in DMEM containing 10% FBS. At 3 d of culture, cells were infected by centrifugation at 2,500 rpm for 2 h at 35 °C with a control retrovirus expressing GFP or with Rgmb- or Rgmb-ΔCtla-4-expressing retrovirus, whose expression can be monitored by IRES-driven expression of GFP, in a solution containing polybrene (2 μg/ml). After infection, the retroviral supernatant was removed and replaced with fresh growth media (20 ng/ml GM-CSF in DMEM containing 10% FBS). At 5 d after infection, non-adherent and loosely adherent cells in the culture supernatant were harvested and stained with PE-Cy7 conjugated anti-mouse CD11c antibodies, then CD11c⁺GFP⁺ cells were sorted as transgene-positive bone marrow derived dendritic cells. Cells were further incubated in 0.1 μg/ml of LPS in DMEM containing 10% FBS for 24 h and supplied for mixed lymphoid reaction with CD8⁺ T cells.

Sekiya T, & Takaki S. (2019). RGMB enhances the suppressive activity of the monomeric secreted form of CTLA-4. Scientific Reports, 9, 6984.

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Stimulatory Factors Modulation

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All-trans-RA, lipopolysaccharide (LPS), cycloheximide, mithramycin A, and phorbol-12-myristate-13-acetate (PMA) were purchased from Sigma-Aldrich. PD98059 and SB203580 were purchased from Merck-Calbiochem. LE540 was gifts from Dr. H. Kagechika (Tokyo Medical and Dental University). Mouse GM-CSF and CpG-ODN1826 were purchased from PeproTech and InvivoGen, respectively.

Ohoka Y., Yokota-Nakatsuma A., Maeda N., Takeuchi H, & Iwata M. (2014). Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells. PLoS ONE, 9(5), e96512.

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OVA and MCCp Peptide Generation

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OVAp-II (OVA 323–339; ISQAVHAAHAEINEAGR) and OVAp-I (OVA 257–264; SIINFEKL) were generated at the Centro de Biología Molecular Severo Ochoa (CBM, Madrid) and Centro Nacional de Biotecnología (CNB-CSIC, Madrid). Moth cytochrome c (MCCp) 88–103 peptide (ANERADLIAYLKQATK) was purchased from GenScript. Other reagents used were mouse GM-CSF (Peprotech), LPS (Sigma-Aldrich), streptavidin microbeads (Miltenyi Biotec), streptavidin-PercP (Becton Dickinson), poly-L-lysine (Sigma-Aldrich), CellTrace Violet, Alexa Fluor568-phalloidin (both from Life Technologies), 7-AAD Viability Staining Solution (eBiosciences), and Live/Dead Fixable dead cell stain (Thermo Fisher). Percp-Streptavidin (1:300 dilution, BD Biosciences), Allophycocyanin-labelled dextramers specific for OVA H-2Kb (257-SIINFEKL-264) were purchased from Immudex.

Cruz-Adalia A., Ramirez-Santiago G., Osuna-Pérez J., Torres-Torresano M., Zorita V., Martínez-Riaño A., Boccasavia V., Borroto A., Martínez del Hoyo G., González-Granado J.M., Alarcón B., Sánchez-Madrid F, & Veiga E. (2017). Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses. Nature Communications, 8, 1591.

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Generation of B11-hCGβ Fusion Protein

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The anti-hMR antibody B11 was generated by immunizing human immunoglobulin transgenic mice with human mannose receptor. The monoclonal antibody (mAb) B11 binds human mannose receptor, but not mouse mannose receptor.^{11 (link)} The B11-hCGβ fusion protein was generated by genetically coupling hCGβ to the carboxyl terminus of the B11 heavy chain, and clinical grade material was manufactured from transfected Chinese hamster ovary cells.^{22 (link),23 (link)} The labeling of B11-hCGβ with Alexa-647 was performed according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Antibodies for staining of CD3ε (145-2C11), CD4 (H129.19), CD8α (53-6.7), CD11c (HL3), MHC class II I-A/I-E (M5/114.15.2), F4/80 (BM8) and CD103 (2E7) were purchased from BD Biosciences (San Jose, CA, USA) or eBioscience (San Diego, CA, USA). hMR was stained with either B11 or 19.2 (BD Biosciences), mouse MR (mMR) with MR5D3, and DEC-205 with NLDC-145 (AbD Serotec, Raleigh, NC, USA and BMA Biomedicals, Augst, Switzerland). Mouse GM-CSF was from Peprotech (Rocky Hill, NJ, USA). Complete Freund's adjuvant (CFA) was from Sigma-Aldrich (St. Louis, MO, USA). CpG (ODN1826) and polyinosinic-polycytidylic acid (poly-IC) were from InvivoGen (San Diego, CA, USA). Poly-ICLC (poly-IC stabilized with poly-lysine and carboxymethylcellulose) was supplied by Oncovir, Inc (Washington, DC, USA).

He L.Z., Weidlick J., Sisson C., Marsh H.C, & Keler T. (2014). Toll-like receptor agonists shape the immune responses to a mannose receptor-targeted cancer vaccine. Cellular and Molecular Immunology, 12(6), 719-728.

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Eosinophil Stimulation and Influenza Virus Infection

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Eosinophils were derived from female C57BL/6 bone marrow for 14 days in eosinophil growth media.^{39 (link)} Sixteen hours before the commencement of the experiment and thereafter, the media was supplemented with mouse GM-CSF (5 ng/ml, PeproTech, Rocky Hill, NJ, USA) to promote the up-regulation of MHC-II. BMdEos were incubated in 0.2 mg/ml A. fumigatus antigen (Greer Labs), or PBS (pH 7.2), for 1 h. Cells were pulsed with a multiplicity of infection (MOI) of 0.1 IAV for 1 h, washed with PBS, and incubated for 24 h in media supplemented with 1 μg/ml TPCK-trypsin (Worthington Biochemical Corporation, Lakewood, NJ, USA). Cells were then transferred to media without TPCK-trypsin and incubated at 37°C/5% CO₂. After 2 days, cells were harvested for flow cytometric staining and analysis.

LeMessurier K.S., Rooney R., Ghoneim H.E., Liu B., Li K., Smallwood H.S, & Samarasinghe A.E. (2020). Influenza A virus directly modulates mouse eosinophil responses. Journal of leukocyte biology, 108(1), 151-168.

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Fabrication and Characterization of MSR Vaccines

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MSRs were fabricated as described previously^{23 (link)}. In brief, 4g of P123 surfactant (average M_n of about 5,800; Sigma-Aldrich) were dissolved in 150 ml of 1.6 M HCl solution and stirred with 8.6 g of tetraethyl-orthosilicate (TEOS, 98%; Sigma-Aldrich) at 40 °C for 20 h, followed by ageing at 100 °C for 24 h. TEOS was extracted in 1% ethanol in HCl at 80 °C for 18 h. To prepare the MSR vaccines, 2 mg of the MSR was adsorbed with 100 μg of murine class B CpG ODN 1826 (sequence 5′-TCCATGACGTTCCTGACGTT-3′; IDT) and 2 mg of MSR was loaded with an equimolar amount of ferritin (control, 120 μg) or 200 μg MICB–ferritin protein. Loading was carried out for 6h at room temperature under shaking, and MSRs were subsequently lyophilized. Separately, 1 mg of the MSRs was loaded with mouse GM-CSF (Peprotech) for 1 h at 37 °C under shaking. MSR vaccines for subsequent boosts were prepared by halving the amounts of protein, CpG and GM-CSF adsorbed to the MSR. The MSRs were combined and resuspended in 150 μl cold PBS before immunization and boost. MSR vaccines were injected via an 18-gauge needle, subcutaneously in the flank region with the mouse under brief isoflurane anaesthesia.

La Teana A., Brandi A., Falconi M., Spurio R., Pon C.L, & Gualerzi C.O. (1991). Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS.. Proceedings of the National Academy of Sciences of the United States of America, 88(23), 10907-10911.

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Dendritic Cell Generation From Mouse Bone Marrow

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Mouse bone marrow (BM) -derived DCs were generated as previously reported^{48 (link)}. Briefly, BM progenitors isolated from the femurs and tibias were incubated at 5 × 10⁵ cells/ml in generating medium (RPMI 1640 containing 10% FBS [Hyclone], 2 mmol/L glutamine, 25 mmol/L HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μmol/L 2-mercaptoethanol and 20 ng/ml of mouse GM-CSF (PeproTech) at 37 °C in a humidified incubator with 5% CO₂ for 6 d to generate immature DCs. Subsequently, immature DCs were incubated in fresh culture medium at 5 × 10⁵ cells/ml in the absence (sham) or presence of HMGN1, R848 (InvivoGen), or LPS (Sigma-Aldrich, as positive control) alone or in combination at specified concentrations for 6–48 h at 37 °C in a humidified incubator with 5% CO₂ before analyzing their function and phenotype. Recombinant HMGN1 used in the present study was generated in insect cells using a baculovirus expressing system and purified as previously described^{27 (link)}.
For the generation of human DCs, peripheral blood monocytes were isolated and cultured in the presence of 50 ng/ml of human GM-CSF (PeproTech) and IL-4 (PeproTech) for 5 to 7 days as previously described^{48 (link)}.

Nie Y., Yang D., Trivett A., Han Z., Xin H., Chen X, & Oppenheim J.J. (2017). Development of a Curative Therapeutic Vaccine (TheraVac) for the Treatment of Large Established Tumors. Scientific Reports, 7, 14186.

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Derivation and Analysis of Atg16l1-Deficient BMDCs

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BMDCs were derived from Atg16l1^f/f and Atg16l1ΔDC mice (Shouval et al., 2014 (link)). BMDCs were cultured in 20 ng/ml mouse GM-CSF (PeproTech, 315-03) for 12 days and were stimulated with 100 ng/ml mouse LPS-EK (InvivoGen, tlrl-eklps) overnight before different assays. Cells were plated on 6-well plates at 1500,000 cells/well overnight and were infected with S. typhimurium at a multiplicity of infection (MOI) of 1:20 for 60 min. After stimulation, cells were lysed in LDS sample buffer (Novex, NP0007), boiled, and added onto SDS-polyacrylamide gels (Invitrogen, NP0341BOX). Proteins were transferred to polyvinylidenedifluoride membranes (Fisher-Scientific, 07-200-165), blocked for 60 min with 1% BSA, and stained overnight with the indicated primary antibody (LC3, Cell Signaling, 3868; P62, Sigma-Aldrich, P0067; b-actin, Cell Signaling, 8457; Phospho-p40phox (Thr154) Antibody, Cell Signaling, 4311; Atg16l1 D6D5, Cell Signaling) at 4 °C. Blots were washed and stained with HRP-conjugated secondary antibody (Goat ant-rabbit IgG-HRP, Santa-Cruz, sc-2004), and binding was detected by chemiluminescence (Thermo Scientific, 34080).

Zhang H., Zheng L., Chen J., Fukata M., Ichikawa R., Shih D.Q, & Zhang X. (2017). The protection role of Atg16l1 in CD11c+ dendritic cells in murine colitis. Immunobiology, 222(7), 831-841.

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Differentiation of Murine Bone Marrow-Derived Dendritic Cells

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The bone marrow cells were freshly isolated from the tibia and femur of 6‐week‐old female C57BL/6 mice and maintained in RPMI‐1640 Medium (Sigma, St. Louis, MO, USA) supplemented with 10% heat‐inactivated FBS (PAA Laboratories, Pasching, Germany), 100 μg·mL⁻¹ streptomycin, and 100 U·mL⁻¹ penicillin (Sigma). To generate immature DCs (iDC), bone marrow cells were cultivated in the first 6 days with mouse GM‐CSF (Peprotech, Rocky Hill, UK, 315‐03) and IL‐4 (Peprotech, 214‐14; 25 ng·mL⁻¹, each).

Horie M., Takagane K., Itoh G., Kuriyama S., Yanagihara K., Yashiro M., Umakoshi M., Goto A., Arita J, & Tanaka M. (2023). Exosomes secreted by ST3GAL5 high cancer cells promote peritoneal dissemination by establishing a premetastatic microenvironment. Molecular Oncology, 18(1), 21-43.

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