Murine m csf

Manufactured by R&D Systems

Sourced in Germany

Murine M-CSF is a recombinant mouse macrophage colony-stimulating factor. It is a hematopoietic growth factor that promotes the survival, proliferation, and differentiation of monocytes and macrophages.

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9 protocols using murine m csf

Isolation and Culture of Immune Cells

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For macrophages culture in vitro, BM cells were isolated from femurs and tibias of 4 to 6‐week‐old WT and Irf8⁻^/⁻ mice, and then passed through a 70 µm cell strainer to obtain single‐cell suspension. After lysed with red blood cell lysis buffer, total BM cells were centrifuged and resuspended at 10⁶ cells mL⁻¹ in culture medium consisting of DMEM (Dulbecco's Modified Eagle's medium), 10% FBS (Fetal Bovine Serum), penicillin, streptomycin, amphotericin and 2‐mercaptoethanol (0.5 mM) supplemented with murine M‐CSF (50 ng mL⁻¹, R&D) for 7 days. On the fourth day of culture, half of the medium was replaced by fresh complete medium. The identity of macrophage was confirmed by flow‐cytometric analysis using fluorochrome conjugated antibodies against CD11b, and F4/80 (BM8).
For NK cell isolation, BM cells were isolated into a single‐cell suspension and stained with biotinylated antibodies against NK1.1 (PK136) at 4 °C for 25 min, then incubated with Streptavidin Microbeads (Miltenyi Biotec) to deplete NK1.1 negative cells. The purity of NK cells was verified by flow‐cytometric analysis using fluorochrome conjugated antibodies against CD3ε, CD49b (DX5) and NK1.1. The same procedure was applied for spleen T cell and B cell isolation, except that the antibody was replaced by biotinylated antibody against CD3ε or CD19.

Li D., Zhang Y., Qiu Q., Wang J., Zhao X., Jiao B., Zhang X., Yu S., Xu P., Dan Y., Xiao X., Wang P., Liu M., Xia Z., Huang Z., Zhang R., Li J., Xie X., Zhang Y., Liu C., Liu P, & Ren R. (2021). IRF8 Impacts Self‐Renewal of Hematopoietic Stem Cells by Regulating TLR9 Signaling Pathway of Innate Immune Cells. Advanced Science, 8(19), 2101031.

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Derivation of Murine and Human Macrophages

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Mouse BMDMs were derived from bone marrow cells of C57BL/6 mice (NCR-Fredrick). Briefly, after lysis of red blood cells, bone marrow cells were cultured in DMEM media containing 10% FBS and 50 ng/ml murine M-CSF (R&D Systems) for 5 days. The cells were then trypsinized and plated for treatment or transfection. Peritoneal macrophages were elicited from C57BL/6 mice by i.p. injection of 1 ml sterile 4% Brewer thioglycollate. Cells were harvested 4 days later by peritoneal lavage and plated on plates. After 1 hour at 37°C, non-adherent cells were removed by washing and adherent macrophages were used for treatment or transfection. Human peripheral blood mononuclear cells (PBMCs) were purchased from ZenBio Inc. PBMCs were cultured in DMEM media containing 10% FBS and 50 ng/ml human M-CSF (R&D Systems) for 5 days. The cells were then trypsinized and plated for treatment or transfection. The animal protocol was approved by the UAB Institutional Animal Care and Use Committee (IACUC).

Xie N., Cui H., Banerjee S., Tan Z., Salomao R., Fu M., Abraham E., Thannickal V.J, & Liu G. (2014). miR-27a regulates inflammatory response of macrophages by targeting IL-10. Journal of immunology (Baltimore, Md. : 1950), 193(1), 327-334.

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Generation and Characterization of Mouse BMDMs

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Mouse BMDMs were derived from bone marrow cells of C57BL/6 mice (NCI-Fredrick). Briefly, bone marrow cells were cultured for 5 days in DMEM media containing 10% FBS and 50 ng/ml murine M-CSF (R&D Systems). The established BMDM were then plated for following experiments. Peritoneal macrophages were elicited by 4% thioglycolate. 4 days after injection, cells were harvested by peritoneal lavage and plated. After 1 hour at 37°C, non-adherent cells were removed and adherent macrophages were used for following experiments. The animal protocol was approved by the UAB Institutional Animal Care and Use Committee (IACUC).

Tan Z., Xie N., Cui H., Moellering D.R., Abraham E., Thannickal V.J, & Liu G. (2015). Pyruvate dehydrogenate kinase 1 participates in macrophage polarization via regulating glucose metabolism. Journal of immunology (Baltimore, Md. : 1950), 194(12), 6082-6089.

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Quantifying Phagocytosis of Bacterial Particles

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For assessment of phagocytosis, 1 × 10⁵ cells were seeded in 12-well plates in standard medium. After >12 hours of settling, cells were incubated for 2 hours with pHRodo Green E. coli or pHRodo Red S. aureus BioParticles (Thermo Fisher Scientific) at a concentration of 1:20. Phagocytosis was performed in phenol-free RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% FCS, 2% HEPES (AppliChem, Darmstadt, Germany), 1% L-glutamine (Invitrogen, Darmstadt, Germany) and 10 pg/mL murine M-CSF (R&D Systems, Minneapolis, MN, USA). Subsequently, cells were washed extensively, and the amount of incorporated BioParticles was evaluated by flow cytometry.

Beneke V., Küster F., Neehus A.L., Hesse C., Lopez-Rodriguez E., Haake K., Rafiei Hashtchin A., Schott J.W., Walter D., Braun A., Wolkers W.F., Ackermann M, & Lachmann N. (2018). An immune cell spray (ICS) formulation allows for the delivery of functional monocyte/macrophages. Scientific Reports, 8, 16281.

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Generation of Murine and Human Macrophages

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To generate murine BM-derived MΦs, BM cells were collected by crushing the hind leg bones in a mortar in FBS-free RPMI-1640. BM cells were collected by passing the suspension through a cell strainer. Red blood cells were lysed, and cells incubated in FBS-free RPMI-1640 for 2 h. Adherent cells were then cultured in RPMI-1640 supplemented with 10% FBS and 5 ng/mL murine M-CSF (R&D Systems, Cat No. 416-ML) for 7 days into MΦs. The MΦs were then co-cultured with 5TGM1 cells for another 48 h to generate MM-MΦs.
To generate human peripheral blood mononuclear cells-derived MΦs, mononuclear cells from healthy donor peripheral blood were collected through Ficoll density gradient centrifugation. They were then incubated in FBS-free RPMI-1640 for 2 hours and adherent cells cultured in RPMI-1640 supplemented with 10% FBS and 10 ng/mL human M-CSF (R&D Systems, Cat No. 216-MC) for 7 days into MΦs. Human MΦs were co-cultured with ARP-1 cells for another 48 h to generate MM-MΦs. Ethical approval to use human samples was granted by the Ethical Committee of West China Hospital, Sichuan University.

Zhang D., Huang J., Wang F., Ding H., Cui Y., Yang Y., Xu J., Luo H., Gao Y., Pan L., Wu Y., Gong Y., Xie L., Liu Z., Qu Y., Zhang L., Liu W., Zhang W., Zhao S., Yi Q., Niu T, & Zheng Y. (2021). BMI1 regulates multiple myeloma-associated macrophage’s pro-myeloma functions. Cell Death & Disease, 12(5), 495.

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Murine Macrophage Generation and Isolation

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Macrophages were generated by plating floating progenitors onto low adherence plates at 0.5-1×10⁶ cells per 3 cm dish in IMDM supplemented with 10% FCS, 100 units/ml Penicillin and 100 µg/ml Streptomycin, 1 mM glutamine, 0.15 mM MTG, 10% M-CSF conditioned media, 5% IL3 conditioned media and 10 ng/ml murine M-CSF (R&D Systems). Macrophages were usually harvested after 7 days by first gently washing the cells with PBS to remove any non-adherent cells and then trypsinizing the adherent layer. Cell purity was checked by FACS for expression of F4/80 (eBioscience 17-4801) and CD11b (eBioscience 12-0112). RNA was prepared by lysing the macrophages in TRIzol.

Gilmour J., Assi S.A., Jaegle U., Kulu D., van de Werken H., Clarke D., Westhead D.R., Philipsen S, & Bonifer C. (2014). A crucial role for the ubiquitously expressed transcription factor Sp1 at early stages of hematopoietic specification. Development (Cambridge, England), 141(12), 2391-2401.

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Bone Marrow Macrophage Differentiation

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Bone marrow derived macrophages (BMDM) were prepared as previously described [31 , 32 ]. Briefly, the bone marrow cells were flushed out using advanced RPMI 1640 (Life Technologies). Cells were differentiated using advanced RPMI 1640 supplemented with 2 mM L-Glutamine, 10% (v/v)) fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin and 50 ng/ml murine M-CSF (R&D System) for 7 days in non-cell culture treated dishes.

Xiang X., Piers T.M., Wefers B., Zhu K., Mallach A., Brunner B., Kleinberger G., Song W., Colonna M., Herms J., Wurst W., Pocock J.M, & Haass C. (2018). The Trem2 R47H Alzheimer’s risk variant impairs splicing and reduces Trem2 mRNA and protein in mice but not in humans. Molecular Neurodegeneration, 13, 49.

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Differentiation of Murine Bone Marrow-Derived Macrophages

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Bone marrow cells were flushed from the femurs of 8-12-week-old mice. Following red blood cell lysis (ACK Lysing Buffer, Gibco), cells were seeded in tissue culture treated dishes and incubated for 3–4 hr. Suspension cells were then pelleted and cultured in RPMI 1640 medium containing 10% FBS, 1% A/A and 20 ng/ml of murine M-CSF (R&D Systems) for 4–6 days to obtain BMDMs.

Holdbrooks A.T., Ankenbauer K.E., Hwang J, & Bellis S.L. (2020). Regulation of inflammatory signaling by the ST6Gal-I sialyltransferase. PLoS ONE, 15(11), e0241850.

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Isolation of Primary Murine Microglia

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Primary murine microglial cultures were prepared as previously described (Fleisher‐Berkovich et al, 2010). Briefly, mixed glial cultures were prepared from the whole brain devoid meninges and cerebellum of P0‐2 mice and cultured in DMEM with Glutamax I (Life Technologies), supplemented with 10% (v/v) FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. About 50% of the medium was replaced every other day, and 10 ng/ml murine M‐CSF (R&D System) was supplemented at day 7 in culture. After 10 days in culture, cells were shaken using horizontal orbital shaker at 200 rpm for 30 min to isolate microglia. The medium containing microglia was pelleted at 200 g for 8 min and reseeded to corresponding plates for experiments. Cell identity was confirmed using anti‐CD68 (Fig 3D) and anti‐CD11b (Fig EV1B) antibodies.

Xiang X., Werner G., Bohrmann B., Liesz A., Mazaheri F., Capell A., Feederle R., Knuesel I., Kleinberger G, & Haass C. (2016). TREM2 deficiency reduces the efficacy of immunotherapeutic amyloid clearance. EMBO Molecular Medicine, 8(9), 992-1004.

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