Murine m csf

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Murine M-CSF is a cell culture reagent that stimulates the proliferation and differentiation of macrophages and their precursors in mice. It is a recombinant form of the mouse colony-stimulating factor 1 (M-CSF) protein.

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88 protocols using murine m csf

Osteoclast Differentiation from Bone Marrow

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Osteoclast differentiation from bone marrow cells has been described previously^{51 (link)}. Briefly, bone marrow cells (2 × 10⁵ cells/well) were cultured in α-minimum essential medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Sigma-Aldrich Inc. St. Louis, MO, USA), 2.2 mg/mL sodium hydrogen carbonate (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and penicillin–streptomycin (50×; FUJIFILM Wako Pure Chemical Corporation) in 24 well plates. Bone marrow cells were cultured with murine M-CSF (20 ng/mL; PeproTech, Cranbury, NJ, USA) for 2 days. For the next 3 days, murine M-CSF (20 ng/mL) and murine RANKL (100 ng/mL; PeproTech) were added.

Omata Y., Tachibana H., Aizaki Y., Mimura T, & Sato K. (2023). Essentiality of Nfatc1 short isoform in osteoclast differentiation and its self-regulation. Scientific Reports, 13, 18797.

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Isolation and Characterization of Murine Bone Marrow Cells

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Primary bone marrow cells were collected from 4- to 8-week old C57BL/6J or hCD68-GFP mice. Bone marrow-derived macrophages (BMMs) were differentiated in vitro from bone marrow flush in α-MEM medium (10% FBS, Pen/Strep, glutamine) with murine M-CSF (30 ng/mL eBioscience) for 6 days. At day 7, macrophages were plated at 2.5 × 10⁵ cells/well in 12-well plates (for efferocytosis assays) or 1.5 × 10⁶ cells/well in 6-well plates (for protein/RNA). Bone marrow stromal cells (BMSCs) were derived from bone marrow flush and cultured in α-MEM medium (20% FBS, Pen/Strep, glutamine) containing 10 nM dexamethasone (Sigma) and used at passage 1–2. Bone marrow neutrophils were isolated as previously described [Swamydas and Lionakis, 2013 (link)]. Briefly, bone marrow was flushed from 8- to 12-week old C57BL/6J mice with RPMI supplemented with 10% FBS and 2 mM EDTA, red blood cells lysed using 0.2% NaCl, and neutrophils separated by density gradient centrifugation (Histopaque 1119 and Histopaque 1077). Neutrophils were harvested at the interface of the Histopaque 1119 and Histopaque 1077 layers and confirmed using FACs anaylsis for CD11b⁺Ly6G⁺ cells.

Michalski M.N., Koh A.J., Weidner S., Roca H, & McCauley L.K. (2016). Modulation of Osteoblastic Cell Efferocytosis by Bone Marrow Macrophages. Journal of cellular biochemistry, 117(12), 2697-2706.

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Differentiation of Bone Marrow Cells into Dendritic Cells and Macrophages

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The femur and tibia from two 6-8 week old C57BL/6 or BALB/c mice (Harlan, Rossdorf, Germany) per experiment were flushed with medium (RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin, 50 µg/ml streptomycin, 100 µg/ml gentamycin; Gibco, Grand Island, New York, USA) to collect bone marrow cells. Erythrocytes were lysed in 150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, Steinheim, Germany), pH 7.2. The remaining bone marrow cells were seeded at 10⁶ cells/ml in medium supplemented with either 5 ng/ml murine GM-CSF (BD Pharmingen, Franklin Lakes, New Jersey, USA) and cultured for 7 days or with 100 ng/ml murine Flt3l (BD Pharmingen, Franklin Lakes, New Jersey, USA) and cultured for 9 days or with 10 ng/ml murine M-CSF (eBioscience Inc., San Diego, California, USA) and cultured for 7 days at 37°C with 5% CO₂ in a humidified atmosphere. The bone marrow-derived cells cultured in the presence of GM-CSF give rise to non-adherent in vitro conventional DC model populations, positive for the marker CD11c. Flt3l promotes the differentiation into a mixed population of in vitro model conventional DCs and plasmacytoid DCs, both positive for CD11c with the plasmacytoid DCs being additionally positive for B220, while the in vitro model MΦs cultured in the presence of M-CSF are positive for CD11b [41] (link)-[43] .

Škrnjug I., Rueckert C., Libanova R., Lienenklaus S., Weiss S, & Guzmán C.A. (2014). The Mucosal Adjuvant Cyclic di-AMP Exerts Immune Stimulatory Effects on Dendritic Cells and Macrophages. PLoS ONE, 9(4), e95728.

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Murine Macrophage Differentiation and Signaling

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Bone marrow from C57BL/6 WT, MyD88, Dectin-1 and TLR9 knockout mice aged 8–10 weeks was harvested and differentiated into macrophages in RPMI containing 10% heat-inactivated FCS, 50 μM β-mercaptoethanol and 40 ng/ml murine M-CSF (eBioscience) for 7 days. The murine macrophage cell line J774A.1 was cultured in RPMI containing 10% heat-inactivated FCS and 50 μM β-mercaptoethanol at 37°C, 5% humified CO₂. siRNAs (Ambion) were transfected using the INTERFERin (Polyplus) transfection reagent according to the manufacturer's instructions. The day before stimulation, macrophages were activated with 200 U/ml recombinant murine IFN-γ (R&D Systems) overnight. Macrophages were treated with FK506 (10 ng/ml, Calbiochem), cytochalasin D (1 or 10 μM, Sigma), the Syk inhibitor piceatannol (50 μM, Sigma), the BTK inhibitor LFM-A13 (25 μM, Sigma), the IKK2 inhibitor SC514 (24 μM, Calbiochem), bafilomycin A1 (100 nM, Sigma) or the TLR9 blocking nucleotide ODN2088 (1 μM, InvivoGen) for 1 h prior to stimulation with swollen conidia at the indicated MOIs, zymosan (50 μg/ml, InvivoGen) or LPS (25 ng/ml, InvivoGen).

Herbst S., Shah A., Mazon Moya M., Marzola V., Jensen B., Reed A., Birrell M.A., Saijo S., Mostowy S., Shaunak S, & Armstrong-James D. (2015). Phagocytosis-dependent activation of a TLR9–BTK–calcineurin–NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus. EMBO Molecular Medicine, 7(3), 240-258.

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Murine Fibrocyte Differentiation Protocol

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Murine fibrocytes were cultured and differentiated as described in detail before (50) . Briefly, mouse splenocytes were isolated by forcing spleens through a 100 µm cell strainer (BD Biosciences, Heidelberg, Germany), collected by centrifugation (300 x g for 5 min) and further purified by erythrocyte lysis. After centrifugation, CD11b-positive monocytes were enriched from spleen cells using CD11b MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. Monocyte-enriched cells were resuspended in Fibrolife basal media (Lifeline Cell Technology, Frederick, USA) supplemented with 10 mM HEPES (Gibco), 2 x non-essential amino acids (Biochrom), 2 mM sodium pyruvate (Biochrom), 4 mM glutamine (Gibco), 100 U/ml penicillin, 100 g/ml streptomycin, 2× ITS-3 (Sigma-Aldrich) and 50 μM 2-mercaptoethanol (Gibco). For fibrocyte differentiation, cells were cultured in the presence of 50 ng/ml murine IL-13 (eBioscience) and 25 ng/ml murine M-CSF (eBioscience). On day 3 of the incubation, wells were further supplemented with a cocktail containing a final concentration of 25 ng/ml IL-13 and 12.5 ng/ml M-CSF in fibrolife media. 5 days later, cells were fixed with 10% neutral buffered formalin and stained with hematoxylin and eosin or cell pellets were snap frozen for RNA extraction.

Rohwer N., Jumpertz S., Erdem M., Egners A., Warzecha K.T., Fragoulis A., Kühl A.A., Kramann R., Neuss S., Rudolph I., Endermann T., Zasada C., Apostolova I., Gerling M., Kempa S., Hughes R., Lewis C.E., Brenner W., Malinowski M.B., Stockmann M., Schomburg L., Faller W., Sansom O.J., Tacke F., Morkel M, & Cramer T. (2019). Non-canonical HIF-1 stabilization contributes to intestinal tumorigenesis. Oncogene, 38(28).

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Isolation and Cultivation of Murine Fibrocytes

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Mouse spleen cells were isolated as described previously, except that the spleens were digested with Accutase (Global Cell Solutions, Charlottesville, VA) for 30 minutes at room temperature according to the manufacturers' instructions [21] (link), [22] (link). Spleen cells were cultured in FibroLife (Lifeline Cell Technology, Frederick, MD) serum-free medium (SFM) including 50 ng/ml murine IL-13 and 25 ng/ml murine M-CSF (PeproTech, Rocky Hill, NJ), as described previously [21] (link), [22] (link), [46] (link). Spleen cells were cultured in flat-bottomed, 96-well, tissue-culture plates at 3.5×10⁵ cells/well in 200 μl with human SAP at the indicated concentrations. On day 3 of the incubation, wells were supplemented with IL-13 and M-CSF, as described previously [21] (link), [22] (link). After 5 days, plates were air-dried, fixed with methanol, and stained with eosin and methylene blue, as described previously [12] –[14] (link), [21] (link), [22] (link), [46] (link). Fibrocytes were counted using the following criteria: an adherent cell with elongated spindle-shaped morphology and an oval nucleus, as described previously [12] –[14] (link), [21] (link), [22] (link), [46] (link). IC50 values were obtained by fitting a sigmoidal dose response curve to the data.

Pilling D, & Gomer R.H. (2014). Persistent Lung Inflammation and Fibrosis in Serum Amyloid P Component (Apcs-/-) Knockout Mice. PLoS ONE, 9(4), e93730.

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Isolation and Alternative Activation of BMDMs

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Bone marrow-derived macrophages (BMDMs) were isolated and cultured as described previously^{15 (link)}. Briefly, bone marrow cells from wild type C57BL/6 mice were isolated from the tibias and femurs and treated with 20 ng/ml of recombinant mouse macrophage colony stimulating factor (Murine M-CSF, PeproTech Canada) for 7 days. After 7 days, bone marrow derived macrophages were treated for either 18 or 30 hours with recombinant IL-4 (20 ng/ml), IL-13 (50 ng/ml), alone, or in combination with OSM (50 ng/ml) or IL-6 (50 ng/ml) (PeproTech Canada). Alternative activation of macrophages was assessed in the cell lysate by measuring arginase-1 protein by western blotting and by arginase-1 and CD206 by flow cytometry. In some instances, BMDMs were lysed and RNA was isolated for NanoString gene expression analysis.

Ayaub E.A., Dubey A., Imani J., Botelho F., Kolb M.R., Richards C.D, & Ask K. (2017). Overexpression of OSM and IL-6 impacts the polarization of pro-fibrotic macrophages and the development of bleomycin-induced lung fibrosis. Scientific Reports, 7, 13281.

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Murine Cell Culture and Cytokine Assays

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Reagents used for cell culture were from Invitrogen (Carlsbad, CA, USA). Reagents for RNA extraction were from Qiagen (Valencia, CA, USA); those for cDNA synthesis and qRT-PCR were from Bio-Rad (Hercules, CA, USA). Murine M-CSF, IFN-γ, IL-4, and IL-10 were from PeproTech (Rocky Hill, NJ, USA). Murine TNF-α was from Thermo Fisher Scientific (Waltham, MA, USA). Azoxymethane and LPS were from Sigma-Aldrich (St. Louis, MO, USA). DSS was from TdB Consultancy (Uppsala, Sweden).

Hardbower D.M., Coburn L.A., Asim M., Singh K., Sierra J.C., Barry D.P., Gobert A.P., Piazuelo M.B., Washington M.K, & Wilson K.T. (2017). EGFR-mediated Macrophage Activation Promotes Colitis-associated Tumorigenesis. Oncogene, 36(27), 3807-3819.

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Hematopoietic Progenitor Cell Differentiation

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Hematopoietic CD41⁺ progenitor cells obtained from iPSC differentiation (8 days after start of EB culture) were seeded at a concentration of 20,000–50,000/ml in basic and complete methylcellulose medium (HSC006 with no cytokines added and HSC007 containing IL-6, erythropoietin, SCF, and IL-3, respectively; R&D Systems). HSC007 was additionally supplemented with 20 ng/ml human G-CSF and 20 ng/ml murine M-CSF, while HSC006 was supplemented with 50 ng/ml murine GM-CSF (all Peprotech). Cells were grown for 7–10 days and colonies composed of more than 50 cells were counted.

Mucci A., Kunkiel J., Suzuki T., Brennig S., Glage S., Kühnel M.P., Ackermann M., Happle C., Kuhn A., Schambach A., Trapnell B.C., Hansen G., Moritz T, & Lachmann N. (2016). Murine iPSC-Derived Macrophages as a Tool for Disease Modeling of Hereditary Pulmonary Alveolar Proteinosis due to Csf2rb Deficiency. Stem Cell Reports, 7(2), 292-305.

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Murine Osteoclastogenesis Induction

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Murine M-CSF, murine TNF and soluble human RANKL were purchased from PeproTech (Rochy Hill, NJ, USA). Recombinant Fibronectin was purchased from Sigma-Aldrich. Alexa Fluor^® 488 phalloidin (A12379) was purchased from Invitrogen (Carlsbad, CA, USA).

Nakano S., Inoue K., Xu C., Deng Z., Syrovatkina V., Vitone G., Zhao L., Huang X.Y, & Zhao B. (2019). G-protein Gα13 functions as a cytoskeletal and mitochondrial regulator to restrain osteoclast function. Scientific Reports, 9, 4236.

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