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Mouse recombinant gm csf

Manufactured by Miltenyi Biotec
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Sourced in Italy, United States

Mouse recombinant GM-CSF is a cytokine protein that stimulates the production and function of granulocytes and macrophages. It is commonly used in laboratory research applications.

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Lab products found in correlation

Celltrace violet dye, by Thermo Fisher Scientific (2 mentions) Cd138 apc, by BioLegend (2 mentions) Recombinant murine gm csf, by Miltenyi Biotec (2 mentions) Live dead fixable blue viability dye, by Thermo Fisher Scientific (2 mentions) Peroxynitrite, by Merck Group (1 mentions) Slide a lyzer dialysis cassette kit, by Thermo Fisher Scientific (1 mentions) Phosphate buffered saline (pbs), by Lonza (1 mentions) Anti cd11b, by Thermo Fisher Scientific (1 mentions) Anti cd11c, by Thermo Fisher Scientific (1 mentions) Anti ki67, by Thermo Fisher Scientific (1 mentions) Anti mhcii, by Thermo Fisher Scientific (1 mentions) Anti foxp3, by Thermo Fisher Scientific (1 mentions) Anti cd4, by Thermo Fisher Scientific (1 mentions) Anti cd80, by Thermo Fisher Scientific (1 mentions) Anti cd86, by Thermo Fisher Scientific (1 mentions) Anti ctla 4, by Thermo Fisher Scientific (1 mentions) Foxp3 transcription factor staining buffer kit, by Cytek Biosciences (1 mentions) Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (1 mentions) Isotype control 2a3, by BioXCell (1 mentions) Recombinant human gm csf, by Miltenyi Biotec (1 mentions)

7 protocols using mouse recombinant gm csf

1

Nitration of Recombinant Mouse GM-CSF

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Recombinant mouse GM-CSF was purchased from Miltenyi Biotech (130-095-742). Cytokine nitration was performed by adding 1mM peroxynitrite (Millipore) to the recombinant protein at 37°C for 15 min in a final volume of 100ul PBS (Lonza). After incubation, the samples were extensively dialyzed in PBS for 5 hours using the Slide-A-Lyzer Dialysis cassette kit, 3,500 MWCO (Thermo Fisher Scientific). Finally, cytokines were collected and used for in vitro assays at 20 ng/ml.
Calì B., Agnellini A.H., Cioccarelli C., Sanchez-Rodriguez R., Predonzani A., Toffolo G.I., Viola A., Bronte V., Arrigoni G., Zonta F., Albertoni L., Mescoli C., Marigo I, & Molon B. (2021). GM-CSF Nitration Is a New Driver of Myeloid Suppressor Cell Activity in Tumors. Frontiers in Immunology, 12, 718098.
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2

OX40L-OX40 Modulation of T Cell Immunity

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Recombinant mouse GM-CSF was purchased from Miltenyi Biotec (Auburn, CA). Recombinant mouse FLT3L was purchased from Gemini Bio-Products (West Sacramento, CA). CellTrace Violet cell proliferation kit was purchased from ThermoFisher Scientific (Waltham, MA). Anti-FOXP3, anti-CD4, anti-OX40L, anti-CD11c, anti-Helios, anti-CTLA4, anti-CD39, anti-CD11b, anti-Sirpα, anti-CD80, anti-CD86, anti-CD274, anti-CD273, anti-MHCII, anti-Ki67, and anti-MGL2 fluorescently coupled antibodies were purchased from ThermoFisher Scientific (Waltham, MA). Foxp3/Transcription Factor Staining Buffer Kit was purchased from Tonbo Biosciences (San Diego, CA). Purified anti-OX40L (RML134L) was purchased from Biolegend (San Diego, CA) and purified anti-OX40 agonist (OX-86) was purchased from ThermoFisher Scientific (Waltham, MA).
Marinelarena A., Bhattacharya P., Kumar P., Maker A.V, & Prabhakar B.S. (2018). Identification of a Novel OX40L+ Dendritic Cell Subset That Selectively Expands Regulatory T cells. Scientific Reports, 8, 14940.
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3

Dendritic Cell-Plasma Cell Coculture

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BM from C57BL/6 WT were used to generate BMDC. BM cells were taken from the tibia and femurs of age matched mice and put into culture in 6 well plates with 20ng/mL recombinant murine GM-CSF (Miltenyi mouse recombinant GM-CSF research grade 130-094-043) for 7 days at a concentration of 1e6 cells/mL, 2mL per well. On day 3 and 5, 1mL was taken from each well and replenished with fresh media with 20ng/mL of GM-CSF. Co-culture experiments: PC were purified from the bone marrow of C57BL/6 or IRF4+/− mice. Co-culture was plated in a 96 well plate at a 1:2 ratio, 20,000 PC with or without 40,000 BMDC in 0.2mL of culture medium/well (10% FBS RPMI) supplemented with 10ng/mL GM-CSF at 37°C with 5% CO2. DC were stained with Cell Trace Violet dye (ThermoFisher) to differentiate from PC. To assess viability, cells were taken directly from culture wells and stained with Live Dead Blue Fixable Viability Dye (Thermo Fisher) and CD138-APC (Biolegend). Viability was assessed both as percentage live and total live cells (based off cell number plated).
Utley A., Chavel C., Lightman S., Holling G.A., Cooper J., Peng P., Liu W., Barwick B.G., Gavile C.M., Maguire O., Murray-Dupuis M., Rozanski C., Jordan M.S., Kambayashi T., Olejniczak S.H., Boise L.H, & Lee K.P. (2020). CD28 Regulates Metabolic Fitness for Long-Lived Plasma Cell Survival. Cell reports, 31(12), 107815.
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4

Dendritic Cell-Plasma Cell Coculture

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BM from C57BL/6 WT were used to generate BMDC. BM cells were taken from the tibia and femurs of age matched mice and put into culture in 6 well plates with 20ng/mL recombinant murine GM-CSF (Miltenyi mouse recombinant GM-CSF research grade 130-094-043) for 7 days at a concentration of 1e6 cells/mL, 2mL per well. On day 3 and 5, 1mL was taken from each well and replenished with fresh media with 20ng/mL of GM-CSF. Co-culture experiments: PC were purified from the bone marrow of C57BL/6 or IRF4+/− mice. Co-culture was plated in a 96 well plate at a 1:2 ratio, 20,000 PC with or without 40,000 BMDC in 0.2mL of culture medium/well (10% FBS RPMI) supplemented with 10ng/mL GM-CSF at 37°C with 5% CO2. DC were stained with Cell Trace Violet dye (ThermoFisher) to differentiate from PC. To assess viability, cells were taken directly from culture wells and stained with Live Dead Blue Fixable Viability Dye (Thermo Fisher) and CD138-APC (Biolegend). Viability was assessed both as percentage live and total live cells (based off cell number plated).
Utley A., Chavel C., Lightman S., Holling G.A., Cooper J., Peng P., Liu W., Barwick B.G., Gavile C.M., Maguire O., Murray-Dupuis M., Rozanski C., Jordan M.S., Kambayashi T., Olejniczak S.H., Boise L.H, & Lee K.P. (2020). CD28 Regulates Metabolic Fitness for Long-Lived Plasma Cell Survival. Cell reports, 31(12), 107815.
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5

Generating Bone Marrow-Derived Dendritic Cells

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BM from both male and female C57BL/6 WT, Ido1−/−, and CD80/CD86−/− mice were used to generate BMDC. BM cells were taken from the tibia and femurs of age matched mice and put into culture in 6 well plates with 20 ng/mL recombinant murine GM-CSF (Miltenyi mouse recombinant GM-CSF research grade 130–094-043) for 7 days at a concentration of 1 × 106 cells/mL, 2mL per well. On day 3 and 5, 1mL was taken from each well and replenished with fresh media + 20 ng/mL of GM-CSF.
Lightman S.M., Peresie J.L., Carlson L.M., Holling G.A., Honikel M.M., Chavel C.A., Nemeth M.J., Olejniczak S.H, & Lee K.P. (2021). Indoleamine 2,3-dioxygenase 1 is Essential for Sustaining Durable Antibody Responses. Immunity, 54(12), 2772-2783.e5.
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6

Immunomodulatory Reagents and Antineoplastic Drugs

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Mouse recombinant GM-CSF, mouse recombinant IL-6, human recombinant GM-CSF, and human recombinant G-CSF were purchased from Miltenyi Biotec (Bologna, Italy). Kb-restricted OVA257–264 peptide (SIINFEKL), was synthesized by JPT (Berlin, Germany). Necrostatin-1, 5Z-7-Oxozeaenol (253863) and TPCA-1 (507475-17-4) were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and pan caspase Inhibitor (Z-VAD-FMK) from R&D Systems (Minneapolis, MN, USA). InVivoMAb anti-mouse PD-1 (RMP1-14) and isotype control (2A3) were purchased from Bioxcell (Lebanon, NH, USA). Chemotherapeutic drugs: 5-fluorouracil (51-21-8); gemcitabine (95058-81-4); docetaxel (114977-28-5); paclitaxel (33069-62-4); oxaliplatin (61825-94-3); cisplatin (15663-27-1), etoposide (33419-42-0), irinotecan (100286-90-6), fludarabine (21679-14-1) and carboplatin (41575-94-4) were purchased by Cayman Chemical (Ann Arbor, MI, USA).
Fiore A., Ugel S., De Sanctis F., Sandri S., Fracasso G., Trovato R., Sartoris S., Solito S., Mandruzzato S., Vascotto F., Hippen K.L., Mondanelli G., Grohmann U., Piro G., Carbone C., Melisi D., Lawlor R.T., Scarpa A., Lamolinara A., Iezzi M., Fassan M., Bicciato S., Blazar B.R., Sahin U., Murray P.J, & Bronte V. (2018). Induction of immunosuppressive functions and NF-κB by FLIP in monocytes. Nature Communications, 9, 5193.
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7

Generating Bone Marrow-Derived Dendritic Cells

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BMDCs were generated as described by Lutz et al. (48 (link)). Briefly, 2 × 106 bone marrow cells from C57BL/6, C3H/HeN, or C3H/HeJ mice were seeded in 10-mm bacteriological culture dishes in RPMI (HyClone, Logan, Utah, USA) medium supplemented with 20 ng/mL mouse recombinant GM-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured for 10 days, after which they were collected via gently pipetting in preparation for incubation with hemocyanins. For most experiments, BMDCs were seeded at 1 × 106 cells/mL in 6-well plates and incubated with the hemocyanins (1 mg/mL each) for 24 h. Then, the supernatants were collected to measure the cytokine levels, whereas the cells were treated with PBS supplemented with 10 mM EDTA and 4 mg/mL lidocaine and washed twice with FACS buffer in preparation for the maturation marker analysis by FACS. LPS from Escherichia coli Serotype R515 (Enzo Life Sciences) and LPS from Salmonella enterica serotype typhimurium (Sigma-Aldrich) and zymosan A (Sigma-Aldrich), were used as positive controls; PBS or unstimulated cells were used as a negative controls. The TLR2 control Pam3CysK4 was from Invitrogen Life Technologies (Waltham, MA, USA). The TLR9 control CpG oligonucleotide was from Oligos (Wilsonville, OR, USA).
Jiménez J.M., Salazar M.L., Arancibia S., Villar J., Salazar F., Brown G.D., Lavelle E.C., Martínez-Pomares L., Ortiz-Quintero J., Lavandero S., Manubens A, & Becker M.I. (2019). TLR4, but Neither Dectin-1 nor Dectin-2, Participates in the Mollusk Hemocyanin-Induced Proinflammatory Effects in Antigen-Presenting Cells From Mammals. Frontiers in Immunology, 10, 1136.
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