Recombinant macrophage colony stimulating factor m csf

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Recombinant macrophage colony-stimulating factor (M-CSF) is a protein that functions as a growth factor for macrophages, a type of immune cell. It is produced using recombinant DNA technology.

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9 protocols using recombinant macrophage colony stimulating factor m csf

Murine Bone Marrow-Derived Macrophage Culture Protocol

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Immortalized murine C57BL/6 bone marrow-derived macrophages (BMDM) were a gift from Doug Golenbock (University of Massachusetts Medical School, Worcester, MA). Macrophages were cultured in complete RPMI medium (cRMPI) (RPMI 1650, l-glutamine, 10% heat-inactivated fetal bovine serum [FBS] [Gibco, Thermo Fisher Scientific, Rockford, IL], 1% penicillin/streptomycin, 1% HEPES buffer, and 50 μM β-mercaptoethanol). Puromycin and blasticidin were added to a final concentration of 5 μg/mL as needed for the selection of transduced cells. Primary BMDMs from C57BL/6 and Dectin-2^−/− mice were harvested as previously described (42 (link)). After harvest, primary BMDMs were grown for 7 days in complete RPMI medium containing 20 ng/mL of recombinant macrophage colony-stimulating factor (M-CSF) (Peprotech, Rocky Hill, NJ) prior to use in assays. HEK293T cells were purchased from the ATCC (American Type Cell Collection, Manassas, VA) and were grown in complete DMEM (DMEM, l-glutamine, 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% HEPES buffer). All cell lines were grown at 37°C in the presence of 5% CO₂.

Reedy J.L., Crossen A.J., Negoro P.E., Harding H.B., Ward R.A., Vargas-Blanco D.A., Timmer K.D., Reardon C.M., Basham K.J., Mansour M.K., Wüthrich M., Fontaine T., Latgé J.P, & Vyas J.M. (2023). The C-Type Lectin Receptor Dectin-2 Is a Receptor for Aspergillus fumigatus Galactomannan. mBio, 14(1), e03184-22.

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Macrophage Polarization Protocols

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The isolated monocytes were cultured for 5 days in RPMI 1640, supplemented with 10% FBS and 25 ng/ml of Recombinant Macrophage Colony-Stimulating Factor (M-CSF, PeproTech, Rocky Hill, USA) to generate M0 macrophages. After 5 days, macrophages were polarized in vitro toward M1 or M2 phenotypes. For M1-like polarization, macrophages were cultured in RPMI-1640 medium supplemented with 100 ng/ml lipopolysaccharides (LPS from E. coli; Sigma-Aldrich) and 50 ng/ml interferon-gamma (IFN-γ; PeproTech) and incubated for 18 h. Meanwhile, for M2-like polarization macrophages were cultured in a complete RPMI-1640 medium supplemented with 20 ng/ml interleukin-4 (IL-4; PeproTech), for 18 h.
The culture medium of M2 macrophages was supplemented with tumor necrosis factor-alpha (TNFα; 20 ng/ml; PeproTech) and Transforming Growth Factor-beta (TGFβ; 10 ng/ml; PeproTech) for 18 h as previously reported [18 (link)].

Soldani C., De Simone G., Polidoro M.A., Morabito A., Franceschini B., Colombo F.S., Anselmo A., Milana F., Lleo A., Torzilli G., Pastorelli R., Donadon M, & Brunelli L. (2024). Riboflavin-LSD1 axis participates in the in vivo tumor-associated macrophage morphology in human colorectal liver metastases. Cancer Immunology, Immunotherapy : CII, 73(4), 63.

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Osteoclast Differentiation Assay

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Culture media, fetal bovine serum, and antibiotics were from Invitrogen (USA), while standard ginsenosides (including Re), anti-β-actin antibody, and TRAP-staining solution were from Sigma (USA). Recombinant macrophage colony stimulating factor (MCSF) was from PeproTech EC (UK). Anti-c-Fos and anti-NFATc1 were from Santa Cruz Biotechnology, Inc. (USA). Recombinant GST-RANKL was purified from an Escherichia coli expression system.

Park C.M., Kim H.M., Kim D.H., Han H.J., Noh H., Jang J.H., Park S.H., Chae H.J., Chae S.W., Ryu E.K., Lee S., Liu K., Liu H., Ahn J.S., Kim Y.O., Kim B.Y, & Soung N.K. (2016). Ginsenoside Re Inhibits Osteoclast Differentiation in Mouse Bone Marrow-Derived Macrophages and Zebrafish Scale Model. Molecules and Cells, 39(12), 855-861.

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Differentiation of Bone Marrow-Derived Macrophages

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BMDMs can be obtained by the differentiation of bone marrow cells, as described previously [14 (link)]. The humerus, femur, and tibia were harvested from 6-week-old male C57BL/6 mice and were sterilised by soaking in 75% alcohol for 10 s. The bone cavity was flushed with cold phosphate-buffered saline (PBS), and the cell suspension was filtered through a 70 μm cell strainer to obtain a single-cell suspension. The harvested cells were seeded in a medium containing 15% foetal bovine serum and 20 ng/mL recombinant macrophage colony-stimulating factor (M-CSF) (Peprotech, Rocky Hill, NJ, USA). After 3 days, half of the medium was replaced with fresh medium, and BMDMs were strongly attached to the plate after 6 days. Flow cytometry (FCM) was performed by the standard methods to identify BMDMs using primary antibodies against F4/80, CD11b, and their isotype controls (Invitrogen, Carlsbad, CA, USA). The expression of F4/80 and CD11b (Abcam, Cambridge, UK) was validated by double immunofluorescence (IF) staining.

Geng R., Lin Y., Ji M., Chang Q., Li Z., Xu L., Zhang W, & Lu J. (2022). MFG-E8 promotes tendon-bone healing by regualting macrophage efferocytosis and M2 polarization after anterior cruciate ligament reconstruction. Journal of Orthopaedic Translation, 34, 11-21.

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Isolation and Differentiation of Fibroblasts and Monocytes

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Fibroblasts were isolated as previously described (10 (link)). Fibroblasts were grown in media containing 10% foetal bovine serum (FBS), sodium 0.87 mM orthopyruvate, 0.87x MEM Non-essential amino acids, 1.75 mM glutamine, 87 U/ml penicillin and 87 ug/ml streptomycin. After 3-4 passages and reaching 80-90% confluence, fibroblasts were detached and used for specific experiments. All cells were at the same passage number for all experiments. Human monocytes from healthy blood donors were obtained from blood cones supplied by the National Blood and Transplant Service (ethical approval ERN_16-0191). Monocytes were isolated by negative selection using RosetteSep Human Monocyte Enrichment Cocktail (STEMCELL Technologies). Cells were then differentiated for 7 days in RPMI 1640 (Thermo Fisher Scientific) supplemented with 5% heat-inactivated FBS in presence of recombinant macrophage colony-stimulating factor (M-CSF) (50 ng/ml; PeproTech) and then used for in vitro experiments.

Pucino V., Nefla M., Gauthier V., Alsaleh G., Clayton S.A., Marshall J., Filer A., Clark A.R., Raza K, & Buckley C.D. (2023). Differential effect of lactate on synovial fibroblast and macrophage effector functions. Frontiers in Immunology, 14, 1183825.

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Isolation and Differentiation of Murine Macrophages

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Bone marrow was isolated from mouse femurs and aliquots prepared in 90% v/v foetal bovine serum (FBS) containing 10% v/v dimethyl sulfoxide (DMSO). Fresh isolated bone marrow was differentiated to macrophages for RNA sequencing studies. Vials were also stored in liquid nitrogen, until required for further confocal and qPCR validation studies. Briefly, bone marrow cells (fresh or frozen) were cultured in vitro in the presence of 50 ng/mL recombinant macrophage colony-stimulating factor (MCSF) (PeproTech; London, UK) to induce differentiation of monocytes to macrophages, as described previously [26 (link)]. For in vitro stimulation, ligands used included Lipid A (Sigma Aldrich; Poole, UK), LPS extracted using modified phenol/water method [27 (link)] from the ileal Crohn’s disease mucosa-associated Escherichia coli isolate, LF82 [28 (link)], or recombinant mouse tumour necrosis factor alpha (TNF) (Catalogue # 315-01A; PeproTech).

Papoutsopoulou S., Morris L., Bayliff A., Mair T., England H., Stagi M., Bergey F., Alam M.T., Sheibani-Tezerji R., Rosenstiel P., Müller W., Martins Dos Santos V.A, & Campbell B.J. (2022). Effects of Human RelA Transgene on Murine Macrophage Inflammatory Responses. Biomedicines, 10(4), 757.

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Macrophage Differentiation and Polarization

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For BMDC differentiation into BM-derived macrophages (BM-Mφs), BMDCs were cultured for an additional 7 days in the same medium supplement with 10 ng/ml recombinant macrophage colony-stimulating factor (M-CSF) (Peprotech). The efficiency of differentiation was confirmed by FACS analysis for CD11b and F4/80 antigen surface expression (Supplementary Fig. S5). To induce the macrophage phenotypic polarization, WEHI274.1 cells and BM-Mφs were treated for 24 hours with 100 ng/ml IFN-γ (Peprotech) or 200 ng/ml LPS (SIGMA) for M1Mφs and 20 ng/ml IL-4 (R&D systems) or 20 ng/ml IL-13 (Miltenyi Biotec) for M2Mφs.

Okubo M., Kioi M., Nakashima H., Sugiura K., Mitsudo K., Aoki I., Taniguchi H, & Tohnai I. (2016). M2-polarized macrophages contribute to neovasculogenesis, leading to relapse of oral cancer following radiation. Scientific Reports, 6, 27548.

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Macrophage Differentiation and Characterization

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All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. All reagents used for cell culture were from Invitrogen (Carlsbad, CA, USA). RNA extraction reagents were from Qiagen (Valencia, CA, USA), and reagents utilized for cDNA synthesis and RT-PCR were from Bio-Rad (Hercules, CA, USA). Recombinant macrophage colony stimulating factor (M-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA). Protease Inhibitor Cocktail, Set III and Phosphatase Inhibitor Cocktail, Set I were purchased from Calbiochem (Darmstadt, Germany). BCA protein assay was from Pierce Biotechnology (Rockford, IL, USA).

Hardbower D.M., Asim M., Murray-Stewart T., Casero RA J.r., Verriere T., Lewis N.D., Chaturvedi R., Piazuelo M.B, & Wilson K.T. (2016). Arginase 2 deletion leads to enhanced M1 macrophage activation and upregulated polyamine metabolism in response to Helicobacter pylori infection. Amino acids, 48(10), 2375-2388.

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Isolation and Cultivation of Murine Bone Marrow Macrophages

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Isolation of murine bone marrow cells and culture of bone marrow-derived macrophages were conducted as previously described (Toda et al. 2021 (link)). Briefly, C57BL/6J male mice were euthanized, and femurs and tibias were separated to flush bone marrow cells in precooled PBS. The isolated cells were incubated in erythrocyte lysis buffer and then washed with PBS. After centrifugation at 200 g for 5 minutes at 4°C, the cells were resuspended in RMPI medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 1% penicillin-streptomycin, and 50 ng/ml recombinant macrophage colony-stimulating factor (M-CSF, PeproTech, USA) and incubated in a cell incubator with 5% CO₂ at 37℃. After 7 days, mouse bone marrow-derived macrophages were harvested and stimulated with murine TGF-β1 (1 ng/ml or 5 ng/ml, PeproTech, USA) and bortezomib (5 nM).

Zhou T., lin L., Zhan Y., Zhang Z., Jiang Y., Wu M., Xue D., Chen L., Weng X, & Huang Z. (2024). Bortezomib restrains M2 polarization and reduces CXCL16-associated CXCR6+CD4 T cell chemotaxis in bleomycin-induced pulmonary fibrosis. Molecular Medicine, 30, 70.

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