Gm csf

Manufactured by InvivoGen

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GM-CSF is a recombinant human granulocyte-macrophage colony-stimulating factor. It is a cytokine that functions in the differentiation and proliferation of granulocytes and macrophages.

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9 protocols using gm csf

Bone-Marrow Derived Macrophage Activation

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Mouse bone-marrow cells were isolated from femurs and tibiae of Csf3r^+/+ mice and cultured at density of 1.5x10⁶ cells/mL in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) 1% L-Glutammine, 1% Pen/Strept, with 20 ng/mL of murine G M-CSF or 25 ng/mL of murine M-CSF (both M-CSF and GM-CSF were purchased from Peprotech). Cells were washed with PBS and medium replaced at day 2 and 5. BMDMs were stimulated on day 7 with murine IFNγ (20 μg/mL) or murine IL-4 (20 μg/mL), alone or in combination with murine G-CSF (50 ng/mL). After 24 hours cells were lysed with Trizol for further mRNA quantification.
For co-culture experiments, GM-CSF-derived BMDMs were stimulated with GM-CSF (50 ng/mL), either alone or in combination with CpG (250 nM) (Invivogen, San Diego, US) or STING agonist cAIMP (1 μg/mL) (Invivogen). In indicated conditions, BMDMs were co-cultured with 3x10⁵ FACS-sorted neutrophils for 24 hours. In transwell experiments neutrophils were added into the upper compartment of a Transwell permeable support with 0.4 μm pore (Corning, NY, US). After 24 hours medium was collected and IL-12p70 concentration was analyzed by ELISA.

Ponzetta A., Carriero R., Carnevale S., Barbagallo M., Molgora M., Perucchini C., Magrini E., Gianni F., Kunderfranco P., Polentarutti N., Pasqualini F., Di Marco S., Supino D., Peano C., Cananzi F., Colombo P., Pilotti S., Alomar S.Y., Bonavita E., Galdiero M.R., Garlanda C., Mantovani A, & Jaillon S. (2019). Neutrophils Driving Unconventional T Cells Mediate Resistance against Murine Sarcomas and Selected Human Tumors. Cell, 178(2), 346-360.e24.

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Plasmacytoid Dendritic Cell Activation

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pDCs were cultured in RPMI 1640 Medium, GlutaMAX (Life Technologies (Thermo Fisher Scientific), Waltham, Ma) containing 10% Fetal Calf Serum (Hyclone (Thermo Fisher Scientific), Waltham, Ma), 100 U/ml Penicillin/Streptomycin (GIBCO (Thermo Fisher Scientific), Waltham, Ma), MEM Non Essential Amino Acids (GIBCO (Thermo Fisher Scientific), Waltham, Ma), and 1mM NA pyruvate (GIBCO (Thermo Fisher Scientific), Waltham, Ma). Cells (1,000,000/mL) were cultured for 24 hours in 96-well flat-bottom plates in the presence of Influenza A/PR/8/34 (H1N1) 82 HA/ml (Charles River Laboratories, Wilmington, MA), PAM3 1 μg/ml and 10 μg/ml (Invivogen, San Diego, CA), 10 ng/mL GM-CSF, 0.1 μg/mL LPS (Invivogen, San Diego, CA), 100 μg/mL heat-killed M. tuberculosis (Invivogen), MOI 1 heat-killed S. aureus (Invivogen, San Diego, CA), and MOI 10 heat-killed L. monocytogenes (Invivogen, San Diego, CA). Blocking experiments were performed by pretreating pDCs 1 hour before stimulation with 1 μM CU-CPT22 (Merck-Millipore, Germany), Human TLR1 Neutralizing antibody—Monoclonal Mouse IgG1 (Invivogen, San Diego, CA), Human TLR2 Detection and Neutralizing antibody—Monoclonal Human IgA2 (Invivogen, San Diego, CA), Mouse IgG1 isotype control antibody (Invivogen, San Diego, CA), Human IgA2 Isotype Control (Invivogen, San Diego, CA). Supernatants were collected after 24-hours of stimulation and frozen until used.

Raieli S., Trichot C., Korniotis S., Pattarini L, & Soumelis V. (2019). TLR1/2 orchestrate human plasmacytoid predendritic cell response to gram+ bacteria. PLoS Biology, 17(4), e3000209.

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Pleural Immune Cell Activation Assay

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Pleural immune cells were cultured in RPMI-1640 GlutaMax supplemented with 10% FCS, 25mM of Hepes, 1 mM sodium pyruvate, 100U/ml of Penicillin-Streptomycin and 20µg/ml of Gentamicin at 37°C in the presence of 5% CO2. Pleural immune cells were stimulated in 12-well plates (106 cells/ml) during 24h by LPS (2µg/ml), Nec-1s (50µM), GM-CSF (20ng/ml) or Trehalose-6,6-dibehenate (TDB, a Mincle agonist) (10µg/ml) (Invivogen). Then supernatants were collected for cytokine measurement. For analyzing the level of phosphorylation of Syk by flow cytometry, PS cells were stimulated during 30min by TDB (10µg/ml).

Bénard A., Podolska M.J., Czubayko F., Kutschick I., Klösch B., Jacobsen A., Naschberger E., Brunner M., Krautz C., Trufa D.I., Sirbu H., Lang R., Grützmann R, & Weber G.F. (2022). Pleural Resident Macrophages and Pleural IRA B Cells Promote Efficient Immunity Against Pneumonia by Inducing Early Pleural Space Inflammation. Frontiers in Immunology, 13, 821480.

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Vaccine Formulation with Adjuvants

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The purified recombinant VP1 protein was adsorbed with Al(OH)₃ (10 mg/ml, Brenntag Biosector, Denmark) at the volume of 1:1, and further formulated with several adjuvants in this study. Adjuvant GIA is composed of GM-CSF (North China Pharmaceutical, China), IFN-α (SinoBiological, China), and Al(OH)₃. CA was composed with CpG1826 (Generay, China) and Al(OH)₃, RA with R848 (Invivogen, USA) and Al(OH)₃, CRA with CpG1826, R848, and Al(OH)₃, MA with MPL (Institute of Medical Engineering, China) and Al(OH)₃, A with Al(OH)₃. Each vaccination contains 10 µg VP1, 500 µg Al(OH)₃ with either of 10 µg GM-CSF and 1 µg IFN-α, or 10 µg CpG1826, or 10 µg R848 (Invivogen, USA), or 50 µg MPL or a combination of 10 µg CpG1826 and 10 µg R848.

Xu D., Jiang S., He Y., Jin X., Zhao G, & Wang B. (2021). Development of a therapeutic vaccine targeting Merkel cell polyomavirus capsid protein VP1 against Merkel cell carcinoma. NPJ Vaccines, 6, 119.

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Generating dendritic cells from mouse bone marrow

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DCs were derived from bone marrow progenitor cells as described.^{32 (link)} Briefly, 6–8 weeks old C57BL/6 female mice were used for the generation of DCs. The bone marrow cells were harvested from the femurs. Then, the cells were cultured in complete RPMI1640 containing mouse recombinant IL-4 (1 ng/ml) and GM-CSF (10 ng/ml) (both Invivogen, ‎San Diego, CA) for 8 days in a petri dish. On day 8, immature DCs were harvested by collecting non-adherent cells and then immediately pulsed by incubation with KPC tumor cell lysates in the presence of 100 ng/ml IFN-γ and 250 ng/ml LPS – E. coli 0111:B4 (both from Invivogen, ‎San Diego, CA). KPC lysates were generated by collecting and resuspending KPC tumor cells at 1 × 10⁶ cells/ml in PBS, followed by irradiation with UV for 20 minutes (0.75 J/cm²) and 24 h incubation.

Yang J., Eresen A., Shangguan J., Ma Q., Yaghmai V, & Zhang Z. (2021). Irreversible electroporation ablation overcomes tumor-associated immunosuppression to improve the efficacy of DC vaccination in a mice model of pancreatic cancer. Oncoimmunology, 10(1), 1875638.

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Nexinhib20 Inhibits Inflammasome Activation

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The stimuli used for treating cells were: GM-CSF (Shenandoah, 200-15), CpG ODN 1826 (InvivoGen, tlrl-1826), CL097 (InvivoGen, tlrl-c97), fMLF (Sigma), PMA (Sigma). The antibodies used for flow cytometry staining are as follows: FITC-conjugated anti-Ly6G (clone 1A8, BD biosciences) and Alexa Fluor 647-conjugated anti-mouse CD11b (clone M1/70, BD biosciences). The anti-Nlrp3 antibody used in this study was from R&D (MAB7578), and detects both human and mouse proteins. Nexinhib20, 4,4-dimethyl-1-(3-nitrophenyl)-2-(1H-1,2,4-triazol-1-yl)pent-1-en-3-one was described previously, its molecular signature and purity were confirmed by mass spectrometry analysis (Johnson et al., 2016 (link)). Nexinhib20 was dissolved at a concentration of 10 mM in DMSO and diluted in PBS. The final concentration of Nexinhib20 in the reaction media was 10 μM.

Johnson J.L., Ramadass M., Haimovich A., McGeough M.D., Zhang J., Hoffman H.M, & Catz S.D. (2017). Increased Neutrophil Secretion Induced by NLRP3 Mutation Links the Inflammasome to Azurophilic Granule Exocytosis. Frontiers in Cellular and Infection Microbiology, 7, 507.

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Dendritic Cell Culture and Activation

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Legs of C57BL/6 mice (Charles River) were
collected. Bone marrow
was washed out onto a 100 μm cell strainer, and cells were washed
and plated in 10 cm Petri dish (Greiner, 633185) in supplemented cell
culture medium as described above. The RPMI medium was additionally
supplemented with 200 ng/mL human Flt3 ligand (Miltenyi Biotec, 130-096-479)
and 5 ng/mL murine GM-CSF (Peprotech, 315-02) and 50 μM β-mercaptoethanol.
Cells were cultured at a density of 15 × 10⁶ per plate
at 10% CO2, 37 °C in humidified atmosphere. After 5 days, medium
was replaced with fresh 200 ng/mL Flt3 ligand and 5 ng/mL murine GM-CSF.
After 9 days, cells were harvested and replated at a density of 3
× 10⁶ cells per plate, and 200 ng/mL fresh Flt3 ligand
and 5 ng/mL GM-CSF containing medium was added up to 10 mL. One day
before T cell coculture, cells were stimulated with 0.3 μg/mL
LPS (Invivogen, vac-3pelps). On the day of T cell coculture, cells
were given 100 ng/mL SIITFEKL peptide (Genscript) for 3 h.

Weiss L., Weiden J., Dölen Y., Grad E.M., van Dinther E.A., Schluck M., Eggermont L.J., van Mierlo G., Gileadi U., Bartoló-Ibars A., Raavé R., Gorris M.A., Maassen L., Verrijp K., Valente M., Deplancke B., Verdoes M., Benitez-Ribas D., Heskamp S., van Spriel A.B., Figdor C.G, & Hammink R. (2023). Direct In Vivo Activation of T Cells with Nanosized Immunofilaments Inhibits Tumor Growth and Metastasis. ACS Nano, 17(13), 12101-12117.

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In Vitro Synthesis of Cytokine mRNAs

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IL-12, IL-27, and GM-CSF plasmids were purchased from InvivoGen (San Diego, CA, USA) and were amplified to generate templates for in vitro transcription. DNA sequences of the cytokines used in this study are listed in supplementary data. mRNA transcripts were synthesized as reported previously^{37 (link), 38 (link)}. mRNAs were synthesized with full substitution of UTP by pseudouridine-5’-triphosphate (TriLink, USA) using AmpliScribe T7-Flash Transcription Kit (Lucigen, USA) following the manufacturer’s instruction. The resulting mRNAs were then purified by RNA Clean & Concentrator (Zymo, USA) and capped using Vaccinia Capping System (NEB, USA) and Cap 2´-O-Methyltransferase (NEB, USA). After one final round of purification, mRNA concentrations were measured using a NanoDrop 2000 Spectrophotometer (ThermoFisher, USA) and stored at −80°C for future use.

Liu J.Q., Zhang C., Zhang X., Yan J., Zeng C., Talebian F., Lynch K., Zhao W., Hou X., Du S., Kang D.D., Deng B., McComb D.W., Bai X.F, & Dong Y. (2022). Intratumoral delivery of IL-12 and IL-27 mRNA using lipid nanoparticles for cancer immunotherapy. Journal of controlled release : official journal of the Controlled Release Society, 345, 306-313.

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Stimulation of Bone Marrow Derived Cells

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Cell suspensions from bone marrow of the indicated genotypes including WT, Myd88^−/− (Adachi et al., 1998 (link)), CD11cΔSyk (Iborra et al., 2012 (link)), Fcer1g^−/− (B6; 129P2-Fcer1g^tm1Rav/J), Clec7a^−/− (Marakalala et al., 2013 (link)), Clec4n^−/− (Saijo et al., 2010 (link)) and Clec4e^−/− (Wells et al., 2008 (link)) were cultured on tissue culture flasks (Falcon Products) in the presence of 20 ng/mL recombinant GM-CSF (Miltenyi Biotec). GM-CSF BM-derived cells were collected on day 6, purified by positive selection with anti-CD11c-microbeads (Miltenyi Biotec) and plated 16 h before stimulation.
In the indicated experiments, GM-CSF BM-derived CD11c⁺ cells (GM-BM, 2x10⁶/mL) were stimulated with LPS EB (100 ng/mL, InvivoGen), plated TDB (1 μg/well, InvivoGen), zymosan (10 μg/mL, InvivoGen) or by co-culture with the indicated ratio of total microbiota from SPF mice, quantified by the optical density at 600 nm (OD_600nm) together with quantification by flow cytometry, in the presence of 100 U/mL Penicillin and 100 μg/mL Streptomycin. Activation of GM-BMs was assessed by quantifying the upregulation of MHCII, CD40, CD86, CCR7 or Syk phosphorylation.
For some experiments, GM-BMs were FACS-sorted based on the expression of MERTK and CD115 into conventional DCs (GM-DCs) and monocyte-derived macrophages (GM-Macs) as previously described (Helft et al., 2015 (link)).

Martínez-López M., Iborra S., Conde-Garrosa R., Mastrangelo A., Danne C., Mann E.R., Reid D.M., Gaboriau-Routhiau V., Chaparro M., Lorenzo M.P., Minnerup L., Saz-Leal P., Slack E., Kemp B., Gisbert J.P., Dzionek A., Robinson M.J., Rupérez F.J., Cerf-Bensussan N., Brown G.D., Bernardo D., LeibundGut-Landmann S, & Sancho D. (2019). Microbiota Sensing by Mincle-Syk Axis in Dendritic Cells Regulates Interleukin-17 and -22 Production and Promotes Intestinal Barrier Integrity. Immunity, 50(2), 446-461.e9.

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