M csf

Manufactured by Merck Group

Sourced in United States, Germany

M-CSF is a recombinant human macrophage colony-stimulating factor. It functions as a growth factor that stimulates the production and differentiation of macrophages from precursor cells.

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Recombinant human M-CSF (10 citations) Macrophage colony stimulating factor (M-CSF) (7 citations) Recombinant M-CSF (5 citations) Human M-CSF (3 citations) Recombinant soluble human M-CSF (2 citations) Recombinant human macrophage colony stimulating factor (M-CSF; (2 citations) Human macrophage-colony stimulating factor (M-CSF) (2 citations) Macrophage colony-stimulatory factor (M-CSF) (2 citations) Rat recombinant M-CSF (2 citations)

146 protocols using m csf

Isolation and Differentiation of Murine Macrophages

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Peritoneal macrophages were isolated by flushing the peritoneal cavity of 10-week-old mice with cold PBS buffer (Dulbecco's PBS without Mg⁺ & Ca²⁺; PAA) and allowed them to become adherent overnight in RPMI medium (RPMI 1640; PAA) supplemented with 10% FCS. Subsequently cells were differentiated in M1 or M2 phenotype by a 4 hours treatment with 1 μg/ml LPS (Sigma-Aldrich) or 10 ng/ml M-CSF (Sigma-Aldrich) respectively [54 (link)].
Bone marrow was isolated by rinsing the shaft of femur and tibia of the same mice with PBS buffer (Dulbecco's PBS without Mg⁺ & Ca²⁺, PAA) and a 27G needle. Hematopoietic stem cell were cultured in DMEM medium (DMEM High glucose; PAA) supplemented with 10% FCS and differentiated in Macrophages with a five days treatment of 50 ng/ml M-CSF (Sigma-Aldrich). Afterwards cells were differentiated in M1 or M2 phenotype by a 48 hours treatment with 1 μg/ml LPS (Sigma-Aldrich) or 10 ng/ml M-CSF (Sigma-Aldrich) respectively [54 (link)].
Same was performed with the macrophage cell line J774A.1 following the previously described protocol. M1 and M2 macrophage phenotype was confirmed by RT-PCR expression analysis of M1 (iNOS and IL-6) and M2 (Arginase and CCR2) specific genes using the previously described protocol and the primers listed in Supplementary Table S6 [55 (link)].

Schmitz R., Valls A.F., Yerbes R., von Richter S., Kahlert C., Loges S., Weitz J., Schneider M., de Almodovar C.R., Ulrich A, & Schmidt T. (2016). TAM receptors Tyro3 and Mer as novel targets in colorectal cancer. Oncotarget, 7(35), 56355-56370.

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Osteoclast Differentiation of Bone Marrow Macrophages

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All animal procedures were approved by the Chosun University Institutional Animal Care and Use Committee (approved number: CICUC2021-S0034). BMMs were isolated from the femurs and tibias of 6-week-old C57BL/6 male mice, as previously described [45 (link)]. Briefly, isolated cells were cultured in α-MEM (Welgene Inc., Gyeongsan, Republic of Korea) supplemented with 10% fetal bovine serum (FBS; Welgene Inc., Gyeongsan, Republic of Korea), 1% penicillin-streptomycin (Welgene Inc., Gyeongsan, Republic of Korea), and 10 ng/mL M-CSF (Sigma-Aldrich, St. Louis, MO, USA) for 16 h. Thereafter, collected nonadherent cells were cultured in 30 ng/mL M-CSF for 48 h. Finally, adherent cells were used as BMM in the present study. To osteoclast differentiation, BMMs were cultured at a density 1 × 10⁴ cells/mL for 72 h in the presence of 30 ng/mL M-CSF and 50 ng/mL RANKL (Sigma-Aldrich, St. Louis, MO, USA) with or without 10, 25, and 50 μg/mL OFLEE.

Seo Y.S., Lim H., Seo J.Y., Kang K.R., Kim D.K., Lee H.H., Oh D.S, & Kim J.S. (2023). The Ethanol Extracts of Osmanthus fragrans Leaves Ameliorate the Bone Loss via the Inhibition of Osteoclastogenesis in Osteoporosis. Plants, 12(2), 253.

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Isolation and Differentiation of Osteoclasts

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Bone marrow cells and hematopoietic cells were extracted from the tibias and femurs of three-week-old BALB/c female mice under general anesthesia, as described previously (19 (link)). Cells were cultured in α-minimum essential medium (α-MEM) with 15% fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin-streptomycin (PS) at 37°C and 5% CO₂ for 3 h to remove adherent cells. Non-adherent cells were seeded onto new plates and cultured in α-MEM with M-CSF (20 ng/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 3 days, which resulted in the growth of bone marrow-derived macrophages (BMMs).
For osteoclast differentiation and TRAP activity staining, BMMs were seeded in 6-well plates and cultured in the presence of M-CSF (20 ng/ml) and RANKL (20 ng/ml; Sigma-Aldrich; Merck KGaA) with or without 1% PRP for 3 days.

Wang D., Weng Y., Guo S., Zhang Y., Zhou T., Zhang M., Wang L, & Ma J. (2017). Platelet-rich plasma inhibits RANKL-induced osteoclast differentiation through activation of Wnt pathway during bone remodeling. International Journal of Molecular Medicine, 41(2), 729-738.

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Retroviral Transduction of Bone Marrow Macrophages

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Retrovirus packaging and BMM infection by using retroviral vectors pMX-IRES-green fluorescent protein (GFP) and pMX-constitutively active (CA)-NFATc1-IRES-GFP were performed as described previously [24 (link)]. In brief, retrovirus packaging was performed by transient transfection of these pMX vectors into Plat-E retroviral packaging cells (Cell Biolabs, San Diego, CA, USA). BMMs were incubated with the viral supernatant from Plat-E cells together with polybrene (6 μg/mL, Sigma-Aldrich) and M-CSF (60 ng/mL) for 12 h. After removing viral supernatant, BMMs were cultured in the presence of M-CSF for 1 day and then treated as indicated.

Ha H., Shim K.S., Kim T., Lee C.J., Park J.H., Kim H.S, & Ma J.Y. (2014). Water extract of the fruits of Alpinia oxyphylla inhibits osteoclast differentiation and bone loss. BMC Complementary and Alternative Medicine, 14, 352.

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Osteoclastogenesis from Mouse Bone Marrow

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Bone marrow was flushed from the long bones of 6–8 week old C57BL/6 WT or DGKζ^−/− mice and cultured in α-minimum Eagle’s medium (α-MEM; Sigma-Aldrich, St. Louis, MO) containing 5% heat-inactivated fetal bovine serum, glutamine and 1/50 volume CMG 14–12 culture supernatant as source of M-CSF (17 ) for 3 days to generate BMMs. For osteoclastogenesis, BMMs were further cultured in the presence of 100 ng/ml GST-RANKL (purified from BL21 with pGEX-6-RANKL plasmid, using HOOK GST protein purification kit, G-BIOSCIENCES, St. Louis, MO) and 25 ng/ml M-CSF (Sigma-Aldrich, St. Louis, MO) for an additional 3–5 days. For TRAP staining, cells were fixed in 4% paraformaldehyde and stained using the leukocyte acid phosphatase kit (Sigma-Aldrich). For bone resorption, BMMs were cultured on tissue culture plates in the presence of osteoclastogenic medium for 2 days, lifted, and replated in equal number on bone slices for additional 2 days. Cells were scraped and bones stained with 20 μg/ml peroxidase-conjugated wheat-germ agglutinin (Sigma-Aldrich) for 1h at room temperature, followed by incubation with 3,3′-diaminobenzidine (Sigma-Aldrich) for 30 min. Bone resorptive pits were analyzed using a light microscope (Nikon) and quantified using Image J software.

Zamani A., Decker C., Cremasco V., Hughes L., Novack D.V, & Faccio R. (2015). Diacylglycerol kinase ζ (DGKζ) is a critical regulator of bone homeostasis via modulation of c-Fos levels in osteoclasts. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 30(10), 1852-1863.

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Infection Assay of Bone Marrow-Derived Macrophages

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Primary wild-type BMDMs from C57BL/6JRj mice (Janvier) were differentiated in DMEM (Sigma) with 20% M-CSF (supernatants of L929 mouse fibroblasts), 10% v/v FCS, 10 mM HEPES, nonessential amino acids and penicillin (100 IU ml⁻¹)/streptomycin (100 μg ml⁻¹) (all BioConcept). One day before infection, BMDMs were seeded into 24- or 96-well plates (Eppendorf) at a density of 1.5 × 10⁵ or 5 × 10⁴ cells per well in DMEM (Sigma) with 10% M-CSF (supernatants of L929 mouse fibroblasts), 10% v/v FCS, 10 mM HEPES and nonessential amino acids (all BioConcept). Where required, BMDMs were pre-stimulated overnight with LPS (from Escherichia coli strain O111:B4 (InvivoGen; tlr-3pelps)). F. novicida were grown overnight at 37 °C with aeration as described above. The bacteria were added to the BMDMs at a multiplicity of infection of 100 or the indicated value. The plates were centrifuged for 5 min at 500g to ensure similar adhesion of the bacteria to the cells and were incubated for 120 min at 37 °C. Next, the medium was replaced with fresh medium containing 10 μg ml⁻¹ gentamicin (BioConcept) to kill extracellular bacteria, then plates were incubated at 37 °C for the indicated length of time.

Brodmann M., Dreier R.F., Broz P, & Basler M. (2017). Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape. Nature Communications, 8, 15853.

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Murine Bone Marrow-Derived Macrophage Generation and Cytokine Stimulation

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For murine BMDMs, bone marrow was flushed from the femur and tibia of mice using ice-cold sterile PBS and the subsequent cell suspension treated with red cell lysis buffer. Treated cells were then washed in ice-cold sterile PBS. BMDMs were generated by incubation of bone marrow cells in RPMI-1640 medium containing 10% FCS and 1X penicillin-streptomycin (both Sigma-Aldrich) (referred to as complete RPMI (cRPMI)) supplemented with 100 ng/ml murine macrophage colony-stimulating factor (M-CSF; Peprotech). M-CSF-supplemented culture medium was replaced on day 3 and adherent BMDMs were harvested on day 5-6.
For cytokine stimulation experiments, plated BMDMs were stimulated with 20 ng/ml murine GM-CSF (Peprotech) or control media (cRPMI supplemented with 20 ng/ml M-CSF) for 16 h. For macrophage-ILC3 co-cultures, 2.5-5 × 10⁴ BMDMs were plates in flat-bottomed 96-well plates (Sigma-Aldrich) in cRPMI supplemented with 20 ng/ml M-CSF overnight before co-culture with flow-sorted intestinal ILC3s (2:1 ratio of macrophages:ILC3s) or ILC3-conditoned media. After 24-72 h, cells were harvested or used for downstream assays. ILC3-conditioned media was generated by culture of ILC3s in cRPMI supplemented with 50 ng/ml murine IL-1β (Peprotech) for 16 h at a concentration of 1 × 10⁶ ILC3s/ml cRPMI. cRPMI supplemented with IL-1β was used as a control in these experiments.

Castro-Dopico T., Fleming A., Dennison T.W., Ferdinand J.R., Harcourt K., Stewart B.J., Cader Z., Tuong Z.K., Jing C., Lok L.S., Mathews R.J., Portet A., Kaser A., Clare S, & Clatworthy M.R. (2020). GM-CSF Calibrates Macrophage Defense and Wound Healing Programs during Intestinal Infection and Inflammation. Cell Reports, 32(1), 107857.

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RNAi Vector Construction and Retroviral Transduction

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RNAi expression vectors were constructed with piGENEmU6 vector (iGENE Therapeutics; Tokyo, Japan) as described [32 (link)] [33 (link)], and the U6 promoter and inserts were cloned into pMx vectors. Retroviruses carrying specific genes were prepared by transfecting BOSC packaging cells with retrovirus vectors and collecting the supernatant after 2 days. For retroviral infection, after the first 2 days of culture, BMMs were incubated with retrovirus in the presence of 30 ng/ml M-CSF and 4 ng/ml polybrene (Sigma-Aldrich; St Louis, MO, USA) for 6 h and cultured overnight in the presence of 10 ng/ml M-CSF. To select the transduced BMMs, cells were detached with Trypsin/EDTA (Sigma-Aldrich; St Louis, MO, USA) and cultured with 10 ng/ml M-CSF and 2 μg/ml puromycin (Sigma-Aldrich) for 2 days. Primer sequences were as follows: shNedd9_1 (sense) 5′-gtttGCATTAGATCTTTGGTCGACAgtgtgctgtccTGTTGGCCAAAGGTCTGATGCttttt-3′, shNedd9_1 (antisense) 5′-atgcaaaaaGCATCAGACCTTTGGCCAACAggacagcacacTGTCGACCAAAGATCTAATGC-3′, shNedd9_2(sense) 5′-gtttGCAGTGCTAGGAGTGACATGTgtgtgctgtccACATGTTACTCCTGGTACTGCttttt-3′, shNedd9_2 (antisense) 5′-atgcaaaaaGCAGTACCAGGAGTAACATGTggacagcacacACATGTCACTCCTAGCACTGC-3′.

Omata Y., Nakamura S., Koyama T., Yasui T., Hirose J., Izawa N., Matsumoto T., Imai Y., Seo S., Kurokawa M., Tsutsumi S., Kadono Y., Morimoto C., Aburatani H., Miyamoto T, & Tanaka S. (2016). Identification of Nedd9 as a TGF-β-Smad2/3 Target Gene Involved in RANKL-Induced Osteoclastogenesis by Comprehensive Analysis. PLoS ONE, 11(6), e0157992.

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Rat Bone Marrow Cell Differentiation

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Bone marrow (BM) cells collected from SD rat femur were cultured in α‐Minimum Essential Medium (α‐MEM) for 3 hours to initiate adherence; then, the non‐adherent cells in the medium were collected and re‐seeded at a density of 1 × 10⁶ cells/mL. The cells were cultured in complete medium with macrophage colony‐stimulating factor (M‐CSF) (20 ng/mL; Sigma Aldrich) for 3 days; then, the medium was refreshed every 3 days with complete medium supplemented with M‐CSF (20 ng/mL) and receptor activator of nuclear factor kappa‐Β ligand (RANKL) (20 ng/mL; Sigma Aldrich).

Tan Q., Wu J., Liu Y., Liu K., Tang J., Ye W., Zhu G., Mei H, & Yang G. (2020). The neurofibromatosis type I gene promotes autophagy via mTORC1 signalling pathway to enhance new bone formation after fracture. Journal of Cellular and Molecular Medicine, 24(19), 11524-11534.

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BMDM Polarization and Gene Expression

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BMCs isolated as described above were seeded at 5 × 10⁶ per 6‐cm dish in RPMI‐1640 (Sigma) with 20% heat‐inactivated FBS (Omega Scientific), penicillin (100 U/ml; Gibco), streptomycin (100 μg/ml; Gibco) and 10 ng/ml M‐CSF (R&D) at 37°C with 5% CO₂ for 6 days. BMDMs were then stimulated for 6‐h with activation media consisting of DMEM with 0.25% heat‐inactivated FBS, penicillin, streptomycin and 10 ng/ml M‐CSF with or without IL‐4 (25 ng/ml; Sigma) and IL‐10 (10 ng/ml; Sigma). RNA was collected in Trizol for QPCR analysis as described above. PPIA and TPT1 were used as reference genes for QPCR experiments using BMDMs.

Wang Y., Welc S.S., Wehling‐Henricks M., Kong Y., Thomas C., Montecino‐Rodriguez E., Dorshkind K, & Tidball J.G. (2022). Myeloid cell‐specific mutation of Spi1 selectively reduces M2‐biased macrophage numbers in skeletal muscle, reduces age‐related muscle fibrosis and prevents sarcopenia. Aging Cell, 21(10), e13690.

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